Patent application title: PICHIA METHANOLICA SECRETORY SIGNAL
Inventors:
Christopher K. Raymond (Seattle, WA, US)
Christopher K. Raymond (Seattle, WA, US)
Michael R. Stamm (Everett, WA, US)
IPC8 Class: AC07K1646FI
USPC Class:
5303873
Class name: Globulins immunoglobulin, antibody, or fragment thereof, other than immunoglobulin antibody, or fragment thereof that is conjugated or adsorbed chimeric, mutated, or recombined hybrid (e.g., bifunctional, bispecific, rodent-human chimeric, single chain, rfv, immunoglobulin fusion protein, etc.)
Publication date: 2009-01-29
Patent application number: 20090030186
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Patent application title: PICHIA METHANOLICA SECRETORY SIGNAL
Inventors:
Christopher K. Raymond
Michael R. Stamm
Agents:
ZYMOGENETICS, INC.;INTELLECTUAL PROPERTY DEPARTMENT
Assignees:
Origin: SEATTLE, WA US
IPC8 Class: AC07K1646FI
USPC Class:
5303873
Abstract:
Novel Pichia methanolica secretory signal polypeptides, polynucleotides
encoding the polypeptides, and related compositions and methods of using
are disclosed. Methods of producing large amounts of recombinant proteins
by employing DNA constructs having a polypeptide of interest preceded by
a novel Pichia methanolica secretory signal sequence.Claims:
1. A fusion protein comprising a first portion and a second portion joined
by a peptide bond, wherein the first portion comprises an amino acid
sequence of SEQ ID NO:2, and the second portion comprises another
polypeptide.
2. The fusion protein of claim 1 wherein the another polypeptide of the second portion comprises an antibody or antibody fragment.
3. A fusion protein comprising a first portion, a second portion, and a third portion, wherein the first portion is joined by a peptide bond to the second portion and the second portion is joined by a peptide bond to the third portion, wherein the first portion comprises an amino acid sequence of SEQ ID NO:2, the second portion comprises another polypeptide, and the third portion comprises an affinity tag, a therapeutic agent or a detectable label.
4. The fusion protein of claim 3 wherein the another polypeptide of the second portion comprises an antibody or antibody fragment.
5. A fusion protein comprising a first portion, a second portion, and a third portion, wherein the first portion is joined by a peptide bond to the second portion and the second portion is joined by a peptide bond to the third portion, wherein the first portion comprises an amino acid sequence of SEQ ID NO:2, the second portion comprises another polypeptide, and the third portion comprises an immunoglobulin moiety.
6. The fusion protein of claim 5 wherein the another polypeptide of the second portion comprises an antibody or antibody fragment.
7. The fusion protein of claim 5 wherein the immunoglobulin moiety of the third portion comprises an immunoglobulin Fc fragment.
Description:
[0001]The present application is a divisional of U.S. patent application
Ser. No. 11/670,807, filed Feb. 2, 2007, which is a divisional of U.S.
patent application Ser. No. 11/170,268, filed Jun. 29, 2005, now U.S.
Pat. No. 7,189,835, which is a divisional of U.S. patent application Ser.
No. 10/903,350, filed Jul. 30, 2004, now U.S. Pat. No. 6,943,234, which
claims the benefit of U.S. Patent Application Ser. Nos. 60/491,093, filed
Jul. 30, 2003, and 60/501,134, filed Sep. 8, 2003, all of which are
herein incorporated by reference.
BACKGROUND OF THE INVENTION
[0002]Methylotrophic yeasts are those yeasts that are able to utilize methanol as a sole source of carbon and energy. Species of yeasts that have the biochemical pathways necessary for methanol utilization are classified in four genera, Hansenula, Pichia, Candida, and Torulopsis. These genera are somewhat artificial, having been based on cell morphology and growth characteristics, and do not reflect close genetic relationships (Billon-Grand, Mycotaxon 35:201-204, 1989; Kurtzman, Mycologia 84:72-76, 1992). Furthermore, not all species within these genera are capable of utilizing methanol as a source of carbon and energy. As a consequence of this classification, there are great differences in physiology and metabolism between individual species of a genus.
[0003]Methylotrophic yeasts are attractive candidates for use in recombinant protein production systems for several reasons. First, some methylotrophic yeasts have been shown to grow rapidly to high biomass on minimal defined media. Second, recombinant expression cassetes are genomically integrated and therefore mitotically stable. Third, these yeasts are capable of secreting large amounts of recombinant proteins. See, for example, Faber et al., Yeast 11:1331, 1995; Romanos et al., Yeast 8:423, 1992; Cregg et al., Bio/Technology 11:905, 1993; U.S. Pat. No. 4,855,242; U.S. Pat. No. 4,857,467; U.S. Pat. No. 4,879,231; and U.S. Pat. No. 4,929,555; and Raymond, U.S. Pat. Nos. 5,716,808, 5,736,383, 5,854,039, and 5,888,768.
[0004]In the commercial production of proteins via recombinant DNA technologies, it is often advantageous for the desired protein of interest to be secreted into the growth medium. Secretion of proteins from cells is generally accomplished by the presence of a short stretch of hydrophobic amino acids constituting the amino-terminal end of the translational product. This hydrophobic stretch is call the "secretory signal sequence," and it is possible to use signal sequences to effect the secretion of heterologous proteins. This is generally accomplished by the construction of an DNA construct comprising a DNA sequence encoding a secretory signal sequence, into which a gene encoding the desired heterologous protein is inserted. When such a plasmid is transformed into a host cell, the host cell will express and secrete the desired protein into the growth medium.
[0005]At present, the only mode of achieving secretion of a heterologous protein product in Pichia methanolica is by way of a foreign secretory signal peptide. Because foreign gene's are not native to Pichia methanolica, the levels of heterologous protein expression are likely suboptimal as compared to a DNA construct incorporating a secretory signal sequence native to Pichia methanolica.
[0006]Thus, there remains a need in the art to identify a secretory signal sequence native to Pichia methanolica to enable the use of methylotrophic yeasts for production of polypeptides of economic importance, including industrial enzymes and pharmaceutical proteins. The present invention provides such materials and methods as well as other, related advantages.
DETAILED DESCRIPTION OF THE INVENTION
[0007]In the description that follows, a number of terms are used extensively. The following definitions are provided to facilitate understanding of the invention.
[0008]Unless otherwise specified, "a," "an," "the," and "at least one" are used interchangeably and mean one or more than one.
[0009]The term "allelic variant" is used herein to denote an alternative form of a gene. Allelic variation is known to exist in populations and arises through mutation.
[0010]A "DNA construct" is a DNA molecule, either single- or double-stranded, that has been modified through human intervention to contain segments of DNA combined and juxtaposed in an arrangement not existing in nature.
[0011]A "DNA segment" is a portion of a larger DNA molecule having specified attributes. For example, a DNA segment encoding a specified polypeptide is a portion of a longer DNA molecule, such as a plasmid or plasmid fragment, that, when read from the 5' to the 3' direction, encodes the sequence of amino acids of the specified polypeptide.
[0012]The term "functionally deficient" denotes the expression in a cell of less than 10% of an activity as compared to the level of that activity in a wild-type counterpart. It is preferred that the expression level be less than 1% of the activity in the wild-type counterpart, more preferably less than 0.01% as determined by appropriate assays. It is most preferred that the activity be essentially undetectable (i.e., not significantly above background). Functional deficiencies in genes can be generated by mutations in either coding or non-coding regions.
[0013]The term "gene" is used herein to denote a DNA segment encoding a polypeptide. Where the context allows, the term includes genomic DNA (with or without intervening sequences), cDNA, and synthetic DNA. Genes may include non-coding sequences, including promoter elements.
[0014]The term "isolated", when applied to a polynucleotide, denotes that the polynucleotide has been removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences, and is in a form suitable for use within genetically engineered protein production systems. Such isolated molecules are those that are separated from their natural environment and include cDNA and genomic clones.
[0015]"Operably linked", when referring to DNA segments, indicates that the segments are arranged so that they function in concert for their intended purposes, e.g., transcription initiates in the promoter and proceeds through the coding segment to the terminator.
[0016]A "polynucleotide" is a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end. Polynucleotides include RNA and DNA, and may be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules. Sizes of polynucleotides are expressed as base pairs (abbreviated "bp"), nucleotides ("nt"), or kilobases ("kb"). Where the context allows, the latter two terms may describe polynucleotides that are single-stranded or double-stranded. When these terms are applied to double-stranded molecules they are used to denote overall length and will be understood to be equivalent to the term "base pairs". It will be recognized by those skilled in the art that the two strands of a double-stranded polynucleotide may differ slightly in length and that the ends thereof may be staggered as a result of enzymatic cleavage; thus all nucleotides within a double-stranded polynucleotide molecule may not be paired. Such unpaired ends will in general not exceed 20 nt in length.
[0017]A "polypeptide" is a polymer of amino acid residues joined by peptide bonds, whether produced naturally or synthetically. Polypeptides of less than about 10 amino acid residues are commonly referred to as "peptides".
[0018]The term "promoter" is used herein for its art-recognized meaning to denote a portion of a gene containing DNA sequences that provide for the binding of RNA polymerase and initiation of transcription. Promoter sequences are commonly, but not always, found in the 5' non-coding regions of genes. Sequences within promoters that function in the initiation of transcription are often characterized by consensus nucleotide sequences. These promoter elements include RNA polymerase binding sites, TATA sequences, and transcription factor binding sites. See, in general, Watson et al., eds., Molecular Biology of the Gene, 4th ed., The Benjamin/Cummings Publishing Company, Inc., Menlo Park, Calif., 1987.
[0019]A "pro sequence" is a DNA sequence that commonly occurs immediately 5' to the mature coding sequence of a gene encoding a secretory protein. The pro sequence encodes a pro peptide that serves as a cis-acting chaperone as the protein moves through the secretory pathway.
[0020]A "protein" is a macromolecule comprising one or more polypeptide chains. A protein may also comprise non-peptidic components, such as carbohydrate groups. Carbohydrates and other non-peptidic substituents may be added to a protein by the cell in which the protein is produced, and will vary with the type of cell. Proteins are commonly defined in terms of their amino acid backbone structures; substituents such as carbohydrate groups are generally not specified, but may be present nonetheless.
[0021]The term "secretory signal sequence" denotes a DNA sequence that encodes a polypeptide (a "secretory peptide") that, as a component of a larger polypeptide, directs the larger polypeptide through a secretory pathway of a cell in which it is synthesized. The larger polypeptide is commonly cleaved to remove the secretory peptide during transit through the secretory pathway. A secretory peptide and a pro peptide may be collectively referred to as a pre-pro peptide.
[0022]As used herein, a "therapeutic agent" is a molecule or atom which is conjugated to an antibody moiety to produce a conjugate which is useful for therapy. Examples of therapeutic agents include drugs, toxins, immunomodulators, chelators, boron compounds, photoactive agents or dyes, and radioisotopes.
[0023]A "detectable label" is a molecule or atom which can be conjugated to an antibody moiety to produce a molecule useful for diagnosis. Examples of detectable labels include chelators, photoactive agents, radioisotopes, fluorescent agents, paramagnetic ions, or other marker moieties.
[0024]The term "affinity tag" is used herein to denote a polypeptide segment that can be attached to a second polypeptide to provide for purification or detection of the second polypeptide or provide sites for attachment of the second polypeptide to a substrate. In principal, any peptide or protein for which an antibody or other specific binding agent is available can be used as an affinity tag. Affinity tags include a poly-histidine tract, protein A (Nilsson et al, EMBO J. 4:1075 (1985); Nilsson et al., Methods Enzymol. 198:3 (1991)), glutathione S transferase (Smith and Johnson, Gene 67:31 (1988)), Glu-Glu affinity tag (Grussenmeyer et al., Proc. Natl Acad. Sci. USA 82:7952 (1985)), substance P, FLAG peptide (Hopp et alt., Biotechnology 6:1204 (1988)), streptavidin binding peptide, or other antigenic epitope or binding domain. See, in general, Ford et al., Protein Expression and Purification 2:95 (1991). Nucleic acid molecules encoding affinity tags are available from commercial suppliers (e.g., Pharmacia Biotech, Piscataway, N.J.).
[0025]All references cited herein are incorporated by reference in their entirety.
[0026]At present, the only mode of achieving secretion of a heterologous protein product in Pichia methanolica is by way of a foreign secretory signal peptide. Because foreign gene's are not native to Pichia methanolica, the levels of heterologous protein expression are likely suboptimal as compared to a DNA construct incorporating a secretory signal sequence native to Pichia methanolica. Without being limited to a theory, a native Pichia methanolica secretory signal peptide would increase heterologous protein production by more effectively directing transport of the heterologous protein to its target membrane, and by being cleaved more efficiently by Pichia methanolica peptidase on the membrane when the heterologous protein passes through it.
[0027]The present invention provides isolated DNA molecules comprising a Pichia methanolica secretory signal sequence, designated exo-1,3-β-glucanase gene and hereinafter referred to as "β-glucanase," is shown in SEQ ID NO:1, the encoded polypeptide is shown in SEQ ID NO:2, and the degenerate DNA molecule encoding the polypeptide of SEQ ID NO:2 is shown in SEQ ID NO:3. Those skilled in the art will recognize that SEQ ID NO:1 represents a single allele of the P. methanolica β-glucanase gene and that other functional alleles (allelic variants) are likely to exist, and that allelic variation may include nucleotide changes. The β-glucanase DNA sequence may be included in a DNA construct. For example, a DNA construct can include the following operably linked elements, which include a Pichia methanolica promoter sequence, β-glucanase DNA sequence, heterologous DNA sequence, and a Pichia methanolica terminator.
[0028]An E. coli DH10B cell culture containing an expression vector encoding Pichia methanolica secretory signal sequence β-glucanase was deposited with the American Type Culture Collection (10801 University Boulevard, Manassas, Va. 20110-2209) on Aug. 1, 2003, and assigned Patent Deposit Designation No. PTA-5369. This deposit will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. The deposit was made merely as a convenience for those of skill in the art and is not an admission that a deposit is required under 35 U.S.C. § 112.
[0029]The present invention provides polynucleotide molecules, including DNA and RNA molecules, which encode the β-glucanase polypeptides disclosed herein. Those skilled in the art will readily recognize that, in view of the degeneracy of the genetic code, considerable sequence variation is possible among these polynucleotide molecules. SEQ ID NO:3 is a degenerate DNA sequence that encompasses all DNAs that encode the β-glucanase polypeptide, and fragments thereof, of SEQ ID NO:2. Those skilled in the art will recognize that the degenerate sequence of SEQ ID NO:3 also provides all RNA sequences encoding SEQ ID NO:2 by substituting U for T. Thus, β-glucanase polypeptide-encoding polynucleotides comprising nucleotide 1 to nucleotide 84 of SEQ ID NO:3 and their RNA equivalents are contemplated by the present invention. Table 1 sets forth the one-letter codes used within SEQ ID NO:3 to denote degenerate nucleotide positions. "Resolutions" are the nucleotides denoted by a code letter. "Complement" indicates the code for the complementary nucleotide(s). For example, the code Y denotes either C or T, and its complement R denotes A or G, with A being complementary to T, and G being complementary to C.
TABLE-US-00001 TABLE 1 Nucleotide Resolution Complement Resolution A A T T C C G G G G C C T T A A R A|G Y C|T Y C|T R A|G M A|C K G|T K G|T M A|C S C|G S C|G W A|T W A|T H A|C|T D A|G|T B C|G|T V A|C|G V A|C|G B C|G|T D A|G|T H A|C|T N A|C|G|T N A|C|G|T
[0030]The degenerate codons used in SEQ ID NO:3, encompassing all possible codons for a given amino acid, are set forth in Table 2.
TABLE-US-00002 TABLE 2 One Degen- Amino Letter erate Acid Code Codons Codon Cys C TGC, TGT TGY Ser S AGC, AGT, TCA, TCC, TCG, TCT WSN Thr T ACA, ACC, ACG, ACT ACN Pro P CCA, CCC, CCG, CCT CCN Ala A GCA, GCC, GCG, GCT GCN Gly G GGA, GGC, GGG, GGT GGN Asn N AAC, AAT AAY Asp D GAC, GAT GAY Glu E GAA, GAG GAR Gln Q CAA, CAG CAR His H CAC, CAT CAY Arg R AGA, AGG, CGA, CGC, CGG, CGT MGN Lys K AAA, AAG AAR Met M ATG ATG Ile I ATA, ATC, ATT ATH Leu L CTA, CTC, CTG, CTT, TTA, TTG YTN Val V GTA, GTC, GTG, GTT GTN Phe F TTC, TTT TTY Tyr Y TAC, TAT TAY Trp W TGG TGG Ter . TAA, TAG, TGA TRR Asn|Asp B RAY Glu|Gln Z SAR Any X NNN
[0031]One of ordinary skill in the art will appreciate that some ambiguity is introduced in determining a degenerate codon, representative of all possible codons encoding each amino acid. For example, the degenerate codon for serine (WSN) can, in some circumstances, encode arginine (AGR), and the degenerate codon for arginine (MGN) can, in some circumstances, encode serine (AGY). A similar relationship exists between codons encoding phenylalanine and leucine. Thus, some polynucleotides encompassed by the degenerate sequence may encode variant amino acid sequences, but one of ordinary skill in the art can easily identify such variant sequences by reference to the amino acid sequence of SEQ ID NO:2. Variant sequences can be readily tested for functionality as described herein.
[0032]A full-length clone encoding β-glucanase can be obtained by conventional cloning procedures. Complementary DNA (cDNA) clones are preferred, although for some applications (e.g., expression in transgenic animals) it may be preferable to use a genomic clone, or to modify a cDNA clone to include at least one genomic intron. Methods for preparing cDNA and genomic clones are well known and within the level of ordinary skill in the art, and include the use of the sequence disclosed herein, or parts thereof, for probing or priming a library. Expression libraries can be probed with antibodies to glucanse fragments, or other specific binding partners.
[0033]The present invention provides an isolated DNA molecule comprising a nucleotide sequence of SEQ ID NO:1 or complement thereof. Those skilled in the art will recognize that the sequence disclosed in SEQ ID NO:1 represents a single allele of human β-glucanase and that allelic variation and alternative splicing are expected to occur. Allelic variants of this sequence can be cloned by probing cDNA or genomic libraries from different individuals according to standard procedures. Allelic variants of the DNA sequence shown in SEQ ID NO:1, including those containing silent mutations and those in which mutations result in amino acid sequence changes, are within the scope of the present invention, as are proteins which are allelic variants of SEQ ID NO:2. cDNAs generated from alternatively spliced mRNAs, which retain the properties of the β-glucanase polypeptide, are included within the scope of the present invention, as are polypeptides encoded by such cDNAs and mRNAs. Allelic variants and splice variants of these sequences can be cloned by probing cDNA or genomic libraries from different individuals or tissues according to standard procedures known in the art.
[0034]The present invention also provides DNA molecules encoding a polypeptide, wherein the encoded polypeptide comprises an amino acid sequence having at least 95 percent sequence identity to SEQ ID NO:2, and wherein the encoded polypeptide is a secretory signal sequence of Pichia methanolica. The polypeptide may comprise, consist essentially of, or consist of SEQ ID NO:2.
[0035]The present invention also provides an isolated polypeptide comprising an amino acid sequence having at least 95 percent sequence identity with SEQ ID NO:2, wherein the polypeptide is a secretory signal sequence of Pichia methanolica. The polypeptide may comprise, consist essentially of, or consist of SEQ ID NO:2.
[0036]The present invention also provides isolated β-glucanase polypeptides that have a substantially similar sequence identity to the polypeptides of SEQ ID NO:2, or their orthologs. The term "substantially similar sequence identity" is used herein to denote polypeptides comprising at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater than 99% sequence identity to the sequences shown in SEQ ID NO:2, or their orthologs. The present invention also includes polypeptides that comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater than 99% sequence identity to the sequence of amino acid residues 1 to 28 of SEQ ID NO:2. The present invention further includes DNA molecules that encode such polypeptides. Methods for determining percent identity are described below.
[0037]The present invention also provides a fusion protein comprising a first portion and a second portion joined by a peptide bond, wherein the first portion comprises an amino acid sequence of SEQ ID NO:2, and the second portion comprises another polypeptide. The second portion may be a heterologous protein to Pichia methanolica. Optionally, a fusion protein of the present invention may further include a third portion which may include, for example, an immuglobulin moiety comprising at least one constant region, e.g., a human immunoglobulin Fc fragment, an affinity tag, a therapeutic agent, a detectable label, and the like.
[0038]The present invention also provides an isolated DNA molecule capable of hybridizing to SEQ ID NO:1, or a complement thereof, under hybridization conditions of 0.015 M NaCl/0.0015 M sodium citrate (SSC) and about 0.1 percent sodium dodecyl sulfate (SDS) at about 50° C. to about 65° C. The nucleic acid molecule may encode at least a portion of a polypeptide, such as a functional β-glucanase of Pichia methanolica.
[0039]The present invention also contemplates variant β-glucanase DNA molecules that can be identified using two criteria: a determination of the similarity between the encoded polypeptide with the amino acid sequence of SEQ ID NO:2, and/or a hybridization assay, as described above. Such β-glucanase variants include nucleic acid molecules: (1) that hybridize with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:1 (or its complement) under stringent washing conditions, in which the wash stringency is equivalent to 0.5×-2× SSC with 0.1% SDS at 55-65° C.; or (2) that encode a polypeptide having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater than 99% identity to the amino acid sequence of SEQ ID NO:2. Alternatively, β-glucanase variants can be characterized as nucleic acid molecules: (1) that hybridize with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:1 (or its complement) under highly stringent washing conditions, in which the wash stringency is equivalent to 0.1×-0.2× SSC with 0.1% SDS at 50-65° C.; and (2) that encode a polypeptide having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater than 99% sequence identity to the amino acid sequence of SEQ ID NO:2.
[0040]Percent sequence identity is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio. 48:603 (1986), and Henikoff and Henikoff, Proc. Natl Acad. Sci. USA 89:10915 (1992). Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the "BLOSUM62" scoring matrix of Henikoff and Henikoff (ibid.) as shown in Table 3 (amino acids are indicated by the standard one-letter codes).
× ##EQU00001##
TABLE-US-00003 TABLE 3 A R N D C Q E G H I L K M F P S T W Y V A 4 R -1 5 N -2 0 6 D -2 -2 1 6 C 0 -3 -3 -3 9 Q -1 1 0 0 -3 5 E -1 0 0 2 -4 2 5 G 0 -2 0 -1 -3 -2 -2 6 H -2 0 1 -1 -3 0 0 -2 8 I -1 -3 -3 -3 -1 -3 -3 -4 -3 4 L -1 -2 -3 -4 -1 -2 -3 -4 -3 2 4 K -1 2 0 -1 -3 1 1 -2 -1 -3 -2 5 M -1 -1 -2 -3 -1 0 -2 -3 -2 1 2 -1 5 F -2 -3 -3 -3 -2 -3 -3 -3 -1 0 0 -3 0 6 P -1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3 -1 -2 -4 7 S 1 -1 1 0 -1 0 0 0 -1 -2 -2 0 -1 -2 -1 4 T 0 -1 0 -1 -1 -1 -1 -2 -2 -1 -1 -1 -1 -2 -1 1 5 W -3 -3 -4 -4 -2 -2 -3 -2 -2 -3 -2 -3 -1 1 -4 -3 -2 11 Y -2 -2 -2 -3 -2 -1 -2 -3 2 -1 -1 -2 -1 3 -3 -2 -2 2 7 V 0 -3 -3 -3 -1 -2 -2 -3 -3 3 1 -2 1 -1 -2 -2 0 -3 -1 4
[0041]Those skilled in the art appreciate that there are many established algorithms available to align two amino acid sequences. The "FASTA" similarity search algorithm of Pearson and Lipman is a suitable protein alignment method for examining the level of identity shared by an amino acid sequence disclosed herein and the amino acid sequence of a putative variant β-glucanase. The FASTA algorithm is described by Pearson and Lipman, Proc. Nat'l Acad. Sci. USA 85:2444 (1988), and by Pearson, Meth. Enzymol. 183:63 (1990).
[0042]Briefly, FASTA first characterizes sequence similarity by identifying regions shared by the query sequence (e.g., SEQ ID NO:2) and a test sequence that have either the highest density of identities (if the ktup variable is 1) or pairs of identities (if ktup=2), without considering conservative amino acid substitutions, insertions, or deletions. The ten regions with the highest density of identities are then rescored by comparing the similarity of all paired amino acids using an amino acid substitution matrix, and the ends of the regions are "trimmed" to include only those residues that contribute to the highest score. If there are several regions with scores greater than the "cutoff" value (calculated by a predetermined formula based upon the length of the sequence and the ktup value), then the trimmed initial regions are examined to determine whether the regions can be joined to form an approximate alignment with gaps. Finally, the highest scoring regions of the two amino acid sequences are aligned using a modification of the Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol Biol 48:444 (1970); Sellers, SIAM J. Appl. Math. 26:787 (1974)), which allows for amino acid insertions and deletions. Preferred parameters for FASTA analysis are: ktup=1, gap opening penalty=10, gap extension penalty=1, and substitution matrix=BLOSUM62. These parameters can be introduced into a FASTA program by modifying the scoring matrix file ("SMATRIX"), as explained in Appendix 2 of Pearson, Meth. Enzymol. 183:63 (1990).
[0043]FASTA can also be used to determine the sequence identity of nucleic acid molecules using a ratio as disclosed above. For nucleotide sequence comparisons, the ktup value can range between one to six, preferably from three to six, most preferably three, with other parameters set as default.
[0044]Variant β-glucanase polypeptides or polypeptides with substantially similar sequence identity are characterized as having one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, that is conservative amino acid substitutions (as shown in Table 4 below) and other substitutions that do not significantly affect the folding or activity of the polypeptide; small deletions, typically of one to about 10 amino acids, preferably one to about 5 amino acids; and amino- or carboxyl-terminal extensions, such as, for instance, an amino-terminal methionine residue, a small linker peptide of up to about 5-20 residues, therapeutic agent, a detectable label, or an affinity tag. The present invention thus includes polypeptides of about 15-100 amino acid residues that comprise a sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater than 99% identical to the corresponding region of SEQ ID NO:2. Polypeptides comprising affinity tags can further comprise a proteolytic cleavage site between the β-glucanase polypeptide and the affinity tag. Preferred such sites include thrombin cleavage sites and factor Xa cleavage sites. Polypeptides of the present invention are preferably recombinant polypeptides. In another aspect, the β-glucanase polypeptides of the present invention have at least 10, at least 15, at least 20, or at least 25 contiguous amino acids. For example, a β-glucanase polypeptide of the present invention relates to a polypeptide having at least 10, at least 15, at least 20, or at least 25 contiguous amino acids of SEQ ID NO:2.
TABLE-US-00004 TABLE 4 Conservative amino acid substitutions Basic: arginine lysine histidine Acidic: glutamic acid aspartic acid Polar: glutamine asparagine Hydrophobic: leucine isoleucine valine Aromatic: phenylalanine tryptophan tyrosine Small: glycine alanine serine threonine methionine
[0045]Determination of amino acid residues that comprise regions or domains that are critical to maintaining structural integrity can be determined. Within these regions one can determine specific residues that will be more or less tolerant of change and maintain the overall tertiary structure of the molecule. Methods for analyzing sequence structure include, but are not limited to, alignment of multiple sequences with high amino acid or nucleotide identity, secondary structure propensities, binary patterns, complementary packing and buried polar interactions (Barton, Current Opin. Struct. Biol 5:372-376, 1995 and Cordes et al., Current Opin. Struct. BioL 6:3-10, 1996). In general, when designing modifications to molecules or identifying specific fragments determination of structure will be accompanied by evaluating activity of modified molecules.
[0046]Amino acid sequence changes are made in β-glucanase polypeptides so as to minimize disruption of higher order structure essential to biological activity. The effects of amino acid sequence changes can be predicted by, for example, computer modeling as disclosed above or determined by analysis of crystal structure (see, e.g., Lapthorn et al., Nat. Struct. Biol. 2:266-268, 1995). Other techniques that are well known in the art compare folding of a variant protein to a standard molecule (e.g., the native protein). For example, comparison of the cysteine pattern in a variant and standard molecules can be made. Mass spectrometry and chemical modification using reduction and alkylation provide methods for determining cysteine residues which are associated with disulfide bonds or are free of such associations (Bean et al., Anal Biochem. 201:216-226, 1992; Gray, Protein Sci. 2:1732-1748, 1993; and Patterson et al., Anal Chem. 66:3727-3732, 1994). It is generally believed that if a modified molecule does not have the same cysteine pattern as the standard molecule folding would be affected. Another well known and accepted method for measuring folding is circular dichrosism (CD). Measuring and comparing the CD spectra generated by a modified molecule and standard molecule is routine (Johnson, Proteins 7:205-214, 1990). Crystallography is another well known method for analyzing folding and structure. Nuclear magnetic resonance (NMR), digestive peptide mapping and epitope mapping are also known methods for analyzing folding and structurally similarities between proteins and polypeptides (Schaanan et al., Science 257:961-964, 1992).
[0047]A Hopp/Woods hydrophilicity profile of the β-glucanase protein sequence as shown in SEQ ID NO:2 can be generated (Hopp et al., Proc. Natl. Acad. Sci., 78:3824-3828, 1981; Hopp, J. Immun. Meth. 88:1-18, 1986 and Triquier et al., Protein Engineering 11:153-169, 1998). The profile is based on a sliding six-residue window. Buried G, S, and T residues and exposed H, Y, and W residues were ignored.
[0048]Those skilled in the art will recognize that hydrophilicity or hydrophobicity will be taken into account when designing modifications in the amino acid sequence of a β-glucanase polypeptide, so as not to disrupt the overall structural and biological profile. Of particular interest for replacement are hydrophobic residues selected from the group consisting of Val, Leu and Ile or the group consisting of Met, Gly, Ser, Ala, Tyr and Trp. For example, residues tolerant of substitution could include Val, Leu and Ile or the group consisting of Met, Gly, Ser, Ala, Tyr and Trp residues as shown in SEQ ID NO:2. Conserved cysteine residues at positions within SEQ ID NO:2 will be relatively intolerant of substitution.
[0049]Using methods such as "FASTA" analysis described previously, regions of high similarity are identified within a family of proteins and used to analyze amino acid sequence for conserved regions. An alternative approach to identifying a variant β-glucanase polynucleotide on the basis of structure is to determine whether a nucleic acid molecule encoding a potential variant β-glucanase gene can hybridize to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:1, as discussed above.
[0050]Other methods of identifying essential amino acids in the polypeptides of the present invention are procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244:1081 (1989), Bass et al., Proc. Natl Acad. Sci. USA 88:4498 (1991), Coombs and Corey, "Site-Directed Mutagenesis and Protein Engineering," in Proteins: Analysis and Design, Angeletti (ed.), pages 259-311 (Academic Press, Inc. 1998)). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological or biochemical activity as disclosed below to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., J. Biol. Chem. 271:4699 (1996).
[0051]The present invention also provides a fusion protein comprising a first portion and a second portion, wherein the first portion and the second portion are joined by a peptide bond, wherein the first portion comprises a functional β-glucanase, such as a polypeptide having at least 95 percent sequence identity with SEQ ID NO:2 or comprising SEQ ID NO:2, and the second portion comprises a protein of interest, such as a heterologous protein. The fusion protein may optionally comprise a third portion, such as an affinity tag, a therapeutic agent, detectable label and the like. The present invention also provides DNA molecules encoding the fusion proteins of the present invention.
[0052]The present invention also provides DNA constructs comprising the following operably linked elements: a first DNA segment comprising a transcription promoter of Pichia methanolica, a second DNA segment comprising a nucleotide sequence encoding a polypeptide of SEQ ID NO:2 or a polypeptide having 95 percent sequence identity with SEQ ID NO:2, a third DNA segment encoding a protein of interest, and a fourth DNA segment comprising a transcription terminator of Pichia methanolica. The first DNA segment may be a transcription promoter such as, for instance, glyceraldehyde-3-phosphate dehydrogenase 1 (GAP1), glyceraldehyde-3-phosphate dehydrogenase 2 (GAP2), alcohol utilization gene 1 (AUG1), alcohol utilization gene 2 (AUG2), and other Pichia methanolica promoters. The second DNA segment is a functional Pichia methanolica β-glucanse gene, e.g., SEQ ID NO:1. The third DNA segment preferably encodes a heterologous protein. The fourth DNA segment includes a Pichia methanolica transcription terminator, such as, for instance, GAP1, GAP2, AUG1, AUG2, and other Pichia methanolica terminators.
[0053]A DNA construct of the present invention may further comprise a selectable marker, e.g., ADE2 gene. In addition, a DNA construct of the present invention may further comprise a Pichia methanolica origin of replication or an additional origin of replication from another organism, e.g., E. coli, Chinese hamster overy (CHO) cells, baby hamster kidney (BHK) cells, and the like. For example, a DNA construct of the present invention can be amplified, for instance, in E. coli then shuttled to a host cell, such as CHO cells, for protein expression.
[0054]A DNA construct of the present invention may further include a fifth operably linked DNA segment wherein the fifth DNA segment comprises an immunoglobulin moiety comprising at least one constant region, for example, a human immunoglobulin Fc fragment, an affinity tag, a therapeutic agent and/or a detectable label.
[0055]Cultured mammalian cells are suitable hosts for DNA constructs of the present invention. Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate-mediated transfection (Wigler et al., Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603, 1981: Graham and Van der Eb, Virology 52:456, 1973), electroporation (Neumann et al., EMBO J. 1:841-5, 1982), DEAE-dextran mediated transfection (Ausubel et al., ibid.), and liposome-mediated transfection (Hawley-Nelson et al., Focus 15:73, 1993; Ciccarone et al., Focus 15:80, 1993, and viral vectors (Miller and Rosman, BioTechniques 7:980-90, 1989; Wang and Finer, Nature Med. 2:714-6, 1996). The production of recombinant polypeptides in cultured mammalian cells is disclosed, for example, by Levinson et al., U.S. Pat. No. 4,713,339; Hagen et al., U.S. Pat. No. 4,784,950; Palmiter et al., U.S. Pat. No. 4,579,821; and Ringold, U.S. Pat. No. 4,656,134. Suitable cultured mammalian cells include the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL 1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) and Chinese hamster ovary (e.g. CHO-K1; ATCC No. CCL 61) cell lines. Additional suitable cell lines are known in the art and available from public depositories such as the American Type Culture Collection, Manassas, Va. In general, strong transcription promoters are preferred, such as promoters from SV-40 or cytomegalovirus. See, e.g., U.S. Pat. No. 4,956,288. Other suitable promoters include those from metallothionein genes (U.S. Pat. Nos. 4,579,821 and 4,601,978) and the adenovirus major late promoter.
[0056]Drug selection is generally used to select for cultured mammalian cells into which foreign DNA has been inserted. Such cells are commonly referred to as "transfectants". Cells that have been cultured in the presence of the selective agent and are able to pass the gene of interest to their progeny are referred to as "stable transfectants." A preferred selectable marker is a gene encoding resistance to the antibiotic neomycin. Selection is carried out in the presence of a neomycin-type drug, such as G-418 or the like. Selection systems can also be used to increase the expression level of the gene of interest, a process referred to as "amplification." Amplification is carried out by culturing transfectants in the presence of a low level of the selective agent and then increasing the amount of selective agent to select for cells that produce high levels of the products of the introduced genes. A preferred amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate. Other drug resistance genes (e.g., hygromycin resistance, multi-drug resistance, puromycin acetyltransferase) can also be used. Alternative markers that introduce an altered phenotype, such as green fluorescent protein, or cell surface proteins such as CD4, CD8, Class I MHC, placental alkaline phosphatase may be used to sort transfected cells from untransfected cells by such means as FACS sorting or magnetic bead separation technology.
[0057]Other higher eukaryotic cells can also be used as hosts, including plant cells, insect cells and avian cells. The use of Agrobacterium rhizogenes as a vector for expressing genes in plant cells has been reviewed by Sinkar et al., J. Biosci. (Bangalore) 11:47-58, 1987. Transformation of insect cells and production of foreign polypeptides therein is disclosed by Guarino et al., U.S. Pat. No. 5,162,222 and WIPO publication No. WO 94/06463. Insect cells can be infected with recombinant baculovirus, commonly derived from Autographa californica nuclear polyhedrosis virus (AcNPV). See, King, L. A. and Possee, R. D., The Baculovirus Expression System: A Laboratory Guide, London, Chapman & Hall; O'Reilly, D. R. et al., Baculovirus Expression Vectors: A Laboratory Manual, New York, Oxford University Press., 1994; and, Richardson, C. D., Ed., Baculovirus Expression Protocols. Methods in Molecular Biology, Totowa, N.J., Humana Press, 1995. The second method of making recombinant baculovirus utilizes a transposon-based system described by Luckow (Luckow, V. A, et al., J Virol 67:4566-79, 1993). This system is sold in the Bac-to-Bac kit (Life Technologies, Rockville, Md.). This system utilizes a transfer vector, pFastBac1® (Life Technologies) containing a Tn7 transposon to move the DNA encoding the β-glucanase fusion protein into a baculovirus genome maintained in E. coli as a large plasmid called a "bacmid." The pFastBac1® transfer vector utilizes the AcNPV polyhedrin promoter to drive the expression of the gene of interest. However, pFastBac1® can be modified to a considerable degree. The polyhedrin promoter can be removed and substituted with the baculovirus basic protein promoter (also known as Pcor, p6.9 or MP promoter) which is expressed earlier in the baculovirus infection, and has been shown to be advantageous for expressing secreted proteins. See, Hill-Perkins, M. S. and Possee, R. D., J. Gen. Virol 71:971-6, 1990; Bonning, B. C. et al., J. Gen. Virol 75:1551-6, 1994; and, Chazenbalk, G. D., and Rapoport, B., J. Biol. Chem. 270:1543-9, 1995. In such transfer vector constructs, a short or long version of the basic protein promoter can be used.
[0058]Using techniques known in the art, a transfer vector containing β-glucanase fusion protein is transformed into E. Coli, and screened for bacmids which contain an interrupted lacZ gene indicative of recombinant baculovirus. The bacmid DNA containing the recombinant baculovirus genome is isolated, using common techniques, and used to transfect Spodoptera frugiperda cells, e.g., Sf9 cells. Recombinant virus that expresses β-glucanase fusion protein is subsequently produced. Recombinant viral stocks are made by methods commonly used the art.
[0059]The recombinant virus is used to infect host cells, typically a cell line derived from the fall armyworm, Spodoptera frugiperda. See, in general, Glick and Pasternak, Molecular Biotechnology: Principles and Applications of Recombinant DNA, ASM Press, Washington, D.C., 1994. Another suitable cell line is the High FiveO® cell line (Invitrogen) derived from Trichoplusia ni (U.S. Pat. No. 5,300,435).
[0060]Fungal cells, including yeast cells, can also be used within the present invention. Yeast species of particular interest in this regard include Saccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica. Methods for transforming S. cerevisiae cells with exogenous DNA and producing recombinant polypeptides therefrom are disclosed by, for example, Kawasaki, U.S. Pat. No. 4,599,311; Kawasaki et al., U.S. Pat. No. 4,931,373; Brake, U.S. Pat. No. 4,870,008; Welch et al., U.S. Pat. No. 5,037,743; and Murray et al., U.S. Pat. No. 4,845,075. Transformed cells are selected by phenotype determined by the selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient (e.g., leucine). A preferred vector system for use in Saccharomyces cerevisiae is the POT1 vector system disclosed by Kawasaki et al. (U.S. Pat. No. 4,931,373), which allows transformed cells to be selected by growth in glucose-containing media. Suitable promoters and terminators for use in yeast include those from glycolytic enzyme genes (see, e.g., Kawasaki, U.S. Pat. No. 4,599,311; Kingsman et al., U.S. Pat. No. 4,615,974; and Bitter, U.S. Pat. No. 4,977,092) and alcohol dehydrogenase genes. See also U.S. Pat. Nos. 4,990,446; 5,063,154; 5,139,936 and 4,661,454. Transformation systems for other yeasts, including Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia guillermondii and Candida maltosa are known in the art. See, for example, Gleeson et al., J. Gen. Microbiol. 132:3459-65, 1986 and Cregg, U.S. Pat. No. 4,882,279. Aspergillus cells may be utilized according to the methods of McKnight et al., U.S. Pat. No. 4,935,349. Methods for transforming Acremonium chrysogenum are disclosed by Sumino et al., U.S. Pat. No. 5,162,228. Methods for transforming Neurospora are disclosed by Lambowitz, U.S. Pat. No. 4,486,533.
[0061]Heterologous or exogenous DNA can also be introduced into P. methanolica cells, another useful yeast host cell, by any of several known methods, including lithium transformation (Hiep et al., Yeast 9:1189-1197, 1993; Tarutina and Tolstorukov, Abst. of the 15th International Specialized Symposium on Yeasts, Riga (USSR), 1991, 137; Ito et al., J. Bacteriol. 153:163, 1983; Bogdanova et al., Yeast 11:343, 1995), spheroplast transformation (Beggs, Nature 275:104, 1978; Hinnen et al., Proc. Natl. Acad. Sci. USA 75:1929, 1978; Cregg et al., Mol. Cell. Biol. 5:3376, 1985), freeze-thaw polyethylene glycol transformation (Pichia Expression Kit Instruction Manual, Invitrogen Corp., San Diego, Calif., Cat. No. K1710-01), or electroporation, the latter being preferred. Electroporation is the process of using a pulsed electric field to transiently permeabilize cell membranes, allowing macromolecules, such as DNA, to pass into cells. Electroporation has been described for use with mammalian (e.g., Neumann et al., EMBO J. 1:841-845, 1982) and fungal (e.g., Meilhoc et al., Bio/Technology 8:223-227, 1990) host cells. However, the actual mechanism by which DNA is transferred into the cells is not well understood. For transformation of P. methanolica, it has been found that electroporation is surprisingly efficient when the cells are exposed to an exponentially decaying, pulsed electric field having a field strength of from 2.5 to 4.5 kV/cm and a time constant (τ) of from 1 to 40 milliseconds. The time constant τ is defined as the time required for the initial peak voltage V0 to drop to a value of V0/e. The time constant can be calculated as the product of the total resistance and capacitance of the pulse circuit, i.e., τ=R×C. Typically, resistance and capacitance are either preset or may be selected by the user, depending on the electroporation equipment selected. In any event, the equipment is configured in accordance with the manufacturer's instructions to provide field strength and decay parameters as disclosed above. Electroporation equipment is available from commercial suppliers (e.g., BioRad Laboratories, Hercules, Calif.).
[0062]Transformed or transfected host cells are cultured according to conventional procedures in a culture medium containing nutrients and other components required for the growth of the chosen host cells. A variety of suitable media, including defined media and complex media, are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins and minerals. Media may also contain such components as growth factors or serum, as required. The growth medium will generally select for cells containing the exogenously added DNA by, for example, drug selection or deficiency in an essential nutrient which is complemented by the selectable marker carried on the expression vector or co-transfected into the host cell. P. methanolica cells, for example, are cultured in a medium comprising adequate sources of carbon, nitrogen and trace nutrients at a temperature of about 25° C. to 35° C. Liquid cultures are provided with sufficient aeration by conventional means, such as shaking of small flasks or sparging of fermentors. A preferred culture medium for P. methanolica is YEPD (2% D-glucose, 2% Bacto® Peptone (Difco Laboratories, Detroit, Mich.), 1% Bacto® yeast extract (Difco Laboratories), 0.004% adenine and 0.006% L-leucine).
[0063]DNA molecules for use in transforming P. methanolica will commonly be prepared as double-stranded, circular plasmids, which are preferably linearized prior to transformation. For polypeptide or protein production, the DNA molecules will include, in addition to the selectable marker disclosed herein, an expression cassette comprising a transcription promoter, a functional glucanase gene, a DNA segment (e.g., a cDNA) encoding the polypeptide or protein of interest, and a transcription terminator. These elements are operably linked to provide for transcription of the DNA segment of interest. It is preferred that the promoter and terminator be that of a P. methanolica gene. Useful promoters include those from constitutive and methanol-inducible promoters. Promoter sequences are generally contained within 1.5 kb upstream of the coding sequence of a gene, often within 1 kb or less. In general, regulated promoters are larger than constitutive promoters due the presence of regulatory elements. Methanol-inducible promoters, which include both positive and negative regulatory elements, may extend more than 1 kb upstream from the initiation ATG. Promoters are identified by function and can be cloned according to known methods.
[0064]A methanol-inducible promoter that may be used is that of a P. methanolica alcohol utilization gene. A representative coding strand sequence of one such gene is AUG1 (Raymond et al., U.S. Pat. No. 6,153,424). P. methanolica contains a second alcohol utilization gene, AUG2, the promoter of which can be used within the present invention (Raymond et al., U.S. Pat. No. 6,153,424). Other useful promoters include those of the dihydroxyacetone synthase (DHAS), formate dehydrogenase (FMD), and catalase (CAT) genes. Genes encoding these enzymes from other species have been described, and their sequences are available (e.g., Janowicz et al., Nuc. Acids Res. 13:2043, 1985; Hollenberg and Janowicz, EPO publication 0 299 108; Didion and Roggenkamp, FEBS Lett. 303:113, 1992). Genes encoding these proteins can be cloned by using the known sequences as probes, or by aligning known sequences, designing primers based on the alignment, and amplifying P. methanolica DNA by the polymerase chain reaction (PCR).
[0065]Constitutive promoters are those that are not activated or inactivated by environmental conditions; they are always transcriptionally active. Preferred constitutive promoters for use within the present invention include those from glyceraldehyde-3-phosphate dehydrogenase (as described herein), triose phosphate isomerase, and phosphoglycerate kinase genes of P. methanolica. These genes can be cloned as disclosed above or by complementation in a host cell, such as a Saccharomyces cerevisiae cell, having a mutation in the counterpart gene. Mutants of this type are well known in the art. See, for example, Kawasaki and Fraenkel, Biochem. Biophys. Res. Comm. 108:1107-1112, 1982; McKnight et al., Cell 46:143-147, 1986; Aguilera and Zimmermann, Mol. Gen. Genet. 202:83-89, 1986.
[0066]The DNA molecule of the present invention can comprise a Pichia methanolica glyceraldehydes-3-phosphate dehydrogenase-1 (GAPDH-1) promoter and terminator (SEQ ID NO:5) (Raymond et al., WO 00/78978), and Pichia methanolica glyceraldehydes-3-phosphate dehydrogenase-2 (GAPDH-2) promoter and terminator (SEQ ID NO:6) (Raymond, U.S. Pat. Nos. 6,348,331 and 6,440,720). For large scale, industrial processes where it is desirable to minimize the use of methanol, host cells may be used that have a genetic defect in a gene required for methanol utilization. Such genes include alcohol oxidase genes AUG1 and AUG2 (Zamost, B., U.S. Pat. No. 6,258,559), as well as genes encoding catalase, formaldehyde dehydrogenase, formate dehydrogenase, dihydroxyacetone synthase, dihydroxyacetone kinase, fructose 1,6-bisphosphate aldolase, and fructose 1,6-bisphosphatase. It is particularly advantageous to use cells in which both alcohol oxidase genes (AUG1 and AUG2) are deleted. Methods for producing Pichia methanolica strains that have a defect in AUG1, AUG2, or both AUG1 and AUG2 genes are described by Raymond et al, Yeast 14:11 (1998), by Raymond, U.S. Pat. No. 5,716,808, and by Raymond et al, U.S. Pat. No. 5,736,383.
[0067]The sequence of a DNA molecule comprising a P. methanolica glyceraldehyde-3-phosphate dehydrogenase-1 (GAPDH-1) gene promoter, coding region, and terminator is shown in SEQ ID NO:5. The gene has been designated GAP1. Those skilled in the art will recognize that SEQ ID NO:5 represents a single allele of the P. methanolica GAP1 gene and that other functional alleles (allelic variants) are likely to exist, and that allelic variation may include nucleotide changes in the promoter region, coding region, or terminator region.
[0068]Within SEQ ID NO:5, the GAP1 open reading frame begins with the methionine codon (ATG) at nucleotides 1733-1735. The transcription promoter is located upstream of the ATG. Gene expression experiments showed that a functional promoter was contained within the ca. 900 nucleotide 5'-flanking region of the GAP1 gene. Analysis of this promoter sequence revealed the presence of a number of sequences homologous to Saccharomyces cerevisiae promoter elements. These sequences include a concensus TATAAA box at nucleotides 1584 to 1591, a consensus Rap1p binding site (Graham and Chambers, Nuc. Acids Res. 22:124-130, 1994) at nucleotides 1355 to 1367, and potential Ger1p binding sites (Shore, Trends Genet. 10:408-412, 1994) at nucleotides 1225 to 1229, 1286 to 1290, 1295 to 1299, 1313 to 1317, 1351 to 1354, 1370 to 1374, 1389 to 1393, and 1457 to 1461. While not wishing to be bound by theory, it is believed that these sequences may perform functions similar to those of their counterparts in the S. cerevisiae TDH3 promoter (Bitter et al., Mol. Gen. Genet. 231:22-32, 1991), that is, they may bind the homologous transcription regulatory elements. Mutation of the region around the consensus Ger1p binding site in the P. methanolica GAP1 promoter has been found to destroy promoter activity.
[0069]Preferred portions of the sequence shown in SEQ ID NO:5 for use within the present invention as transcription promoters include segments comprising at least 900 contiguous nucleotides of the 5' non-coding region of SEQ ID NO:5, and preferably comprising nucleotide 810 to nucleotide 1724 of the sequence shown in SEQ ID NO:5. Those skilled in the art will recognize that longer portions of the 5' non-coding region of the P. methanolica GAP1 gene can also be used. Promoter sequences of the present invention can thus include the sequence of SEQ ID NO:5 through nucleotide 1732 in the 3' direction and can extend to or beyond nucleotide 232 in the 5' direction. For convenience and ease of manipulation, the promoter used within an expression DNA construct will generally not exceed 1.5 kb in length, and will often not exceed 1.0 kb in length.
[0070]As disclosed in more detail in the examples that follow, the sequence of SEQ ID NO:5 from nucleotide 810 to 1724 provides a functional transcription promoter. However, additional nucleotides can be removed from either or both ends of this sequence and the resulting sequence tested for promoter function by joining it to a sequence encoding a protein, preferably a protein for which a convenient assay is readily available.
[0071]Within the present invention it is preferred that the GAP1 promoter be substantially free of GAP1 gene coding sequence, which begins with nucleotide 1733 in SEQ ID NO: 1. As used herein, the term "substantially free of GAP1 gene coding sequence" means that the promoter DNA includes not more than 15 nucleotides of the GAP1 coding sequences, preferably not more than 10 nucleotides, and more preferably not more than 3 nucleotides. Within one embodiment of the invention, the GAP1 promoter is provided free of coding sequence of the P. methanolica GAP1 gene. However, those skilled in the art will recognize that a GAP1 gene fragment that includes the initiation ATG (nucleotides 1733 to 1735) of SEQ ID NO:5 can be operably linked to a heterologous coding sequence that lacks an ATG, with the GAP1 ATG providing for initiation of translation of the heterologous sequence. Those skilled in the art will further recognize that additional GAP1 coding sequences can also be included, whereby a fusion protein comprising GAP1 and heterologous amino acid sequences is produced. Such a fusion protein may comprise a cleavage site to facilitate separation of the GAP1 and heterologous sequences subsequent to translation.
[0072]In addition to the GAP1 promoter sequence, the present invention also provides transcription terminator sequences derived from the 3' non-coding region of the P. methanolica GAP1 gene. A consensus transcription termination sequence (Chen and Moore, Mol. Cell. Biol. 12:3470-3481, 1992) is at nucleotides 2774 to 2787 of SEQ ID NO:5. Within the present invention, there are thus provided transcription terminator gene segments of at least about 60 bp in length. Longer segments, for example at least 90 bp in length or about 200 bp in length, will often be used. These segments comprise the termination sequence disclosed above, and may have as their 5' termini nucleotide 2735 of SEQ ID NO:5. Those skilled in the art will recognize, however, that the transcription terminator segment that is provided in an DNA construct can include at its 5' terminus the TAA translation termination codon at nucleotides 2732-2734 of SEQ ID NO:5 to permit the insertion of coding sequences that lack a termination codon.
[0073]The present invention also provides a DNA molecule comprising a Pichia methanolica glyceraldehyde-3-phosphate dehydrogenase-2 (GAPDH-2) gene promoter, coding region, and terminator as shown in SEQ ID NO:6. The gene has been designated GAP2. Those skilled in the art will recognize that SEQ ID NO:6 represents a single allele of the P. methanolica GAP2 gene and that other functional alleles (allelic variants) are likely to exist, and that allelic variation may include nucleotide changes in the promoter region, coding region, or terminator region.
[0074]Within SEQ ID NO:6, the GAP2 open reading frame begins with the methionine codon (ATG) at nucleotides 1093-1095. The transcription promoter is located upstream of the ATG. Gene expression experiments showed that a functional promoter was contained within the ca. 1000 nucleotide 5'-flanking region of the GAP2 gene.
[0075]Preferred portions of the sequence shown in SEQ ID NO:6 for use within the present invention as transcription promoters include segments comprising at least 900 contiguous nucleotides of the 5' non-coding region of SEQ ID NO:6, and preferably comprising nucleotide 93 to nucleotide 1080 of the sequence shown in SEQ ID NO:6. Those skilled in the art will recognize that longer portions of the 5' non-coding region of the P. methanolica GAP2 gene can also be used. Promoter sequences of the present invention can thus include the sequence of SEQ ID NO:6 through nucleotide 1092 in the 3' direction and can extend to or beyond nucleotide 1 in the 5' direction. In general, the promoter used within an expression DNA construct will not exceed 1.5 kb in length, and will preferably not exceed 1.0 kb in length. In addition to these promoter fragments, the invention also provides isolated DNA molecules of up to about 3300 bp, as well as isolated DNA molecules of up to 5000 bp, wherein said molecules comprise the P. methanolica GAP2 promoter sequence.
[0076]Within the present invention it is preferred that the GAP2 promoter be substantially free of GAP2 gene coding sequence, which begins with nucleotide 1093 in SEQ ID NO:6. As used herein, "substantially free" of GAP2 gene coding sequence means that the promoter DNA includes not more than 15 nucleotides of the GAP2 coding sequence, preferably not more than 10 nucleotides, and more preferably not more than 3 nucleotides. Within a preferred embodiment of the invention, the GAP2 promoter is provided free of coding sequence of the P. methanolica GAP2 gene. However, those skilled in the art will recognize that a GAP2 gene fragment that includes the initiation ATG (nucleotides 1093 to 1095) of SEQ ID NO:6 can be operably linked to a heterologous coding sequence that lacks an ATG, with the GAP2 ATG providing for initiation of translation of the heterologous sequence. Those skilled in the art will further recognize that additional GAP2 coding sequences can also be included, whereby a fusion protein comprising GAP2 and heterologous amino acid sequences is produced. Such a fusion protein may comprise a cleavage site to facilitate separation of the GAP2 and heterologous sequences subsequent to translation.
[0077]In addition to the GAP2 promoter sequence, the present invention also provides transcription terminator sequences derived from the 3' non-coding region of the P. methanolica GAP2 gene. A consensus transcription termination sequence (Chen and Moore, Mol. Cell. Biol. 12:3470-3481, 1992) is at nucleotides 2136 to 2145 of SEQ ID NO:6. Within the present invention, there are thus provided transcription terminator gene segments of at least about 50 bp, preferably at least 60 bp, more preferably at least 90 bp, still more preferably about 200 bp in length. The terminator segments of the present invention may comprise 500-1000 nucleotides of the 3' non-coding region of SEQ ID NO:6. These segments comprise the termination sequence disclosed above, and preferably have as their 5' termini nucleotide 2095 of SEQ ID NO:6. Those skilled in the art will recognize, however, that the transcription terminator segment that is provided in an expression vector can include at its 5' terminus the TAA translation termination codon at nucleotides 2092-2094 of SEQ ID NO:6 to permit the insertion of coding sequences that lack a termination codon.
[0078]A DNA construct of the present invention may further include a selectable marker. Expression vectors or DNA constructs of the present invention further comprise a selectable marker to permit identification and selection of P. methanolica cells containing the vector. Selectable markers provide for a growth advantage of cells containing them. The general principles of selection are well known in the art. The selectable marker is preferably a P. methanolica gene. Commonly used selectable markers are genes that encode enzymes required for the synthesis of amino acids or nucleotides. Cells having mutations in these genes cannot grow in media lacking the specific amino acid or nucleotide unless the mutation is complemented by the selectable marker. Use of such "selective" culture media ensures the stable maintenance of the heterologous DNA within the host cell. A selectable marker of the present invention for use in P. methanolica may include, for instance, a P. methanolica ADE2 gene, which encodes phosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC 4.1.1.21). See, Raymond, U.S. Pat. No. 5,736,383. The ADE2 gene, when transformed into an ade2 host cell, allows the cell to grow in the absence of adenine. The coding strand of a representative P. methanolica ADE2 gene sequence is shown in SEQ ID NO:4. The sequence illustrated includes 1006 nucleotides of 5' non-coding sequence and 442 nucleotides of 3' non-coding sequence, with the initiation ATG codon at nucleotides 1007-1009. Within a preferred embodiment of the invention, a DNA segment comprising nucleotides 407-2851 is used as a selectable marker, although longer or shorter segments could be used as long as the coding portion is operably linked to promoter and terminator sequences. In the alternative, a dominant selectable marker, which provides a growth advantage to wild-type cells, may be used. Typical dominant selectable markers are genes that provide resistance to antibiotics, such as neomycin-type antibiotics (e.g., G418), hygromycin B, and bleomycin/phleomycin-type antibiotics (e.g., Zeocin®; available from Invitrogen Corporation, San Diego, Calif.). A preferred dominant selectable marker for use in P. methanolica is the Sh bla gene, which inhibits the activity of Zeocin®.
[0079]The present invention also provides a Pichia methanolica cell containing a DNA construct as described herein. The DNA construct may be genomically integrated into the Pichia methanolica genome with one or more copies. The Pichia methanolica cell may have a functionally deficient vacuolar proteinease A and/or vacuolar proteinase B. The Pichia methanolica cell may have a functionally deficient AUG1 and/or AUG2 gene.
[0080]The present invention also provides a method of producing a protein of interest comprising: culturing a cell of the present invention wherein the cell containing a DNA construct of the present invention wherein the third DNA segment is expressed and the protein of interest is produced, and recovering the protein of interest. Preferably, the protein of interest is heterologous or foreign to Pichia methanolica.
[0081]Techniques for manipulating cloned DNA molecules and introducing exogenous DNA into a variety of host cells are well known in the art and are disclosed by, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; Murray, ed., Gene Transfer and Expression Protocols, Humana Press, Clifton, N.J., 1991; Glick and Pasternak, Molecular Biotechnology: Principles and Applications of Recombinant DNA, ASM Press, Washington, D.C., 1994; Ausubel et al. (eds.), Short Protocols in Molecular Biology, 3rd edition, John Wiley and Sons, Inc., NY, 1995; Wu et al., Methods in Gene Biotechnology, CRC Press, New York, 1997. DNA vectors, including expression vectors, commonly contain a selectable marker and origin of replication that function in a bacterial host (e.g., E. coli) to permit the replication and amplification of the vector in a prokaryotic host. If desired, these prokaryotic elements can be removed from a vector before it is introduced into an alternative host. For example, such prokaryotic sequences can be removed by linearization of the vector prior to its introduction into a P. methanolica host cell.
[0082]Within other embodiments of the invention, DNA constructs are provided that comprise a DNA segment comprising a portion of SEQ ID NO:6 that is a functional transcription terminator operably linked to a functional β-glucanase gene of the present invention, and an additional DNA segment encoding a protein of interest. Within one embodiment, the GAP2 promoter and terminator sequences of the present invention are used in combination, wherein both are operably linked to a functional β-glucanase gene and a DNA segment encoding a protein of interest within a DNA construct.
[0083]The use of P. methanolica cells as a host for the production of recombinant proteins is disclosed in U.S. Pat. Nos. 5,955,349, 5,888,768, 6,001,597, 5,965,389, 5,736,383, 5,854,039, 5,716,808, 5,736,383, 5,854,039, and 5,736,383. DNA constructs, e.g., expression vectors, for use in transforming P. methanolica will commonly be prepared as double-stranded, circular plasmids, which are preferably linearized prior to transformation. To facilitate integration of the expression vector DNA into the host chromosome, it is preferred to have the entire expression segment of the plasmid flanked at both ends by host DNA sequences (e.g., AUG1 3' sequences). Electroporation is used to facilitate the introduction of a plasmid containing DNA encoding a polypeptide of interest into P. methanolica cells. It is preferred to transform P. methanolica cells by electroporation using an exponentially decaying, pulsed electric field having a field strength of from 2.5 to 4.5 kV/cm, preferably about 3.75 kV/cm, and a time constant (τ) of from 1 to 40 milliseconds, most preferably about 20 milliseconds.
[0084]Integrative transformants are preferred for use in protein production processes. Such cells can be propagated without continuous selective pressure because DNA is rarely lost from the genome. Integration of DNA into the host chromosome can be confirmed by Southern blot analysis. Briefly, transformed and untransformed host DNA is digested with restriction endonucleases, separated by electrophoresis, blotted to a support membrane, and probed with appropriate host DNA segments. Differences in the patterns of fragments seen in untransformed and transformed cells are indicative of integrative transformation. Restriction enzymes and probes can be selected to identify transforming DNA segments (e.g., promoter, terminator, heterologous DNA, and selectable marker sequences) from among the genomic fragments.
[0085]Differences in expression levels of heterologous proteins can result from such factors as the site of integration and copy number of the expression cassette among individual isolates. It is therefore advantageous to screen a number of isolates for expression level prior to selecting a production strain. Isolates exhibiting a high expression level will commonly contain multiple integrated copies of the desired expression cassette. A variety of suitable screening methods are available. For example, transformant colonies are grown on plates that are overlayed with membranes (e.g., nitrocellulose) that bind protein. Proteins are released from the cells by secretion or following lysis, and bind to the membrane. Bound protein can then be assayed using known methods, including immunoassays. More accurate analysis of expression levels can be obtained by culturing cells in liquid media and analyzing conditioned media or cell lysates, as appropriate. Methods for concentrating and purifying proteins from media and lysates will be determined in part by the protein of interest. Such methods are readily selected and practiced by the skilled practitioner.
[0086]For production of secreted proteins, host cells having functional deficiencies in the vacuolar proteases proteinase A, which is encoded by the PEP4 gene, and proteinase B, which is encoded by the PRB1 gene, are preferred in order to minimize spurious proteolysis (Raymond et al., U.S. Pat. No. 6,153,424). Vacuolar protease activity (and therefore vacuolar protease deficiency) is measured using any of several known assays. Preferred assays are those developed for Saccharomyces cerevisiae and disclosed by Jones, Methods Enzymol 194:428-453, 1991. A preferred such assay is the APNE overlay assay, which detects activity of carboxypeptidase Y (CpY). See, Wolf and Fink, J. Bact. 123:1150-1156, 1975. Because the zymogen (pro)CpY is activated by proteinase A and proteinase B, the APNE assay is indicative of vacuolar protease activity in general. The APNE overlay assay detects the carboxypeptidase Y-mediated release of β-naphthol from N-acetyl-phenylalanine-β-naphthyl-ester (APNE), which results in the formation of an isoluble red dye by the reaction of the β-naphthol with the diazonium salt Fast Garnet GBC. Cells growing on assay plates (YEPD plates are preferred) at room temperature are overlayed with 8 ml R×M. R×M is prepared by combining 0.175 g agar, 17.5 ml H2O, and 5 ml 1 M Tris-HCl pH 7.4, microwaving the mixture to dissolve the agar, cooling to ˜55° C., adding 2.5 ml freshly made APNE (2 mg/ml in dimethylformamide) (Sigma Chemical Co., St. Louis, Mo.), and, immediately before assay, 20 mg Fast Garnet GBC salt (Sigma Chemical Co.). The overlay is allowed to solidify, and color development is observed. Wild-type colonies are red, whereas CPY deletion strains are white. Carboxypeptidase Y activity can also be detected by the well test, in which cells are distributed into wells of a microtiter test plate and incubated in the presence of N-benzoyl-L-tyrosine p-nitroanilide (BTPNA) and dimethylformamide. The cells are permeabilized by the dimethylformamide, and CpY in the cells cleaves the amide bond in the BTPNA to give the yellow product p-nitroaniline. Assays for CpY will detect any mutation that reduces protease activity so long as that activity ultimately results in the reduction of CpY activity.
[0087]P. methanolica cells are cultured in a medium comprising adequate sources of carbon, nitrogen and trace nutrients at a temperature of about 25° C. to 35° C. Liquid cultures are provided with sufficient aeration by conventional means, such as shaking of small flasks or sparging of fermentors. A preferred culture medium for P. methanolica is YEPD (2% D-glucose, 2% Bacto® Peptone (Difco Laboratories, Detroit, Mich.), 1% Bacto® yeast extract (Difco Laboratories), 0.004% adenine, 0.006% L-leucine).
[0088]For large-scale culture, one to two colonies of a P. methanolica strain can be picked from a fresh agar plate (e.g., YEPD agar) and suspended in 250 ml of YEPD broth contained in a two-liter baffled shake flask. The culture is grown for 16 to 24 hours at 30° C. and 250 rpm shaking speed. Approximately 50 to 80 milliliters of inoculum are used per liter starting fermentor volume (5-8% v/v inoculum).
[0089]A preferred fermentation medium is a soluble medium comprising glucose as a carbon source, inorganic ammonia, potassium, phosphate, iron, and citric acid. As used herein, a "soluble medium" is a medium that does not contain visible precipitation. Preferably, the medium lacks phosphate glass (sodium hexametaphosphate). A preferred medium is prepared in deionized water and does not contain calcium sulfate. As a minimal medium, it is preferred that the medium lacks polypeptides or peptides, such as yeast extracts. However, acid hydrolyzed casein (e.g., casamino acids or amicase) can be added to the medium if desired. An illustrative fermentation medium is prepared by mixing the following compounds: (NH4)2SO4 (11.5 grams/liter), K2HPO4 (2.60 grams/liter), KH2PO4 (9.50 grams/liter), FeSO4.7H2O (0.40 grams/liter), and citric acid (1.00 gram/liter). After adding distilled, deionized water to one liter, the solution is sterilized by autoclaving, allowed to cool, and then supplemented with the following: 60% (w/v) glucose solution (47.5 milliliters/liter), 10× trace metals solution (20.0 milliliters/liter), 1 M MgSO4 (20.0 milliliters/liter), and vitamin stock solution (2.00 milliliters/liter). The 10× trace metals solution contains FeSO4.7H2O (100 mM), CuSO4.5H2O (2 mM), ZnSO4.7H2O (8 mM), MnSO4.H2O (8 mM), CoCl2.6H2O (2 mM), Na2MoO4.2H2O (1 mM), H3BO3 (8 mM), KI (0.5 mM), NiSO4.6H2O (1 mM), thiamine (0.50 grams/liter), and biotin (5.00 milligrams/liter). The vitamin stock solution contains inositol (47.00 grams/liter), pantothenic acid (23.00 grams/liter), pyrodoxine (1.20 grams/liter), thiamine (5.00 grams/liter), and biotin (0.10 gram/liter). Those of skill in the art can vary these particular ingredients and amounts. For example, ammonium sulfate can be substituted with ammonium chloride, or the amount of ammonium sulfate can be varied, for example, from about 11 to about 22 grams/liter.
[0090]After addition of trace metals and vitamins, the pH of the medium is typically adjusted to pH 4.5 by addition of 10% H3PO4. Generally, about 10 milliliters/liter are added, and no additional acid addition will be required. During fermentation, the pH is maintained between about 3.5 to about 5.5, or about 4.0 to about 5.0, depending on protein produced, by addition of 5 N NH4OH.
[0091]An illustrative fermentor is a BIOFLO 3000 fermentor system (New Brunswick Scientific Company, Inc.; Edison, N.J.). This fermentor system can handle either a six-liter or a fourteen-liter fermentor vessel. Fermentations performed with the six-liter vessel are prepared with three liters of medium, whereas fermentations performed with the fourteen-liter vessel are prepared with six liters of medium. The fermentor vessel operating temperature is typically set to 30° C. for the course of the fermentation, although the temperature can range between 27-31° C. depending on the protein expressed. The fermentation is initiated in a batch mode. The glucose initially present is often used by approximately 10 hours elapsed fermentation time (EFT), at which time a glucose feed can be initiated to increase the cell mass. An illustrative glucose feed contains 900 milliliters of 60% (w/v) glucose, 60 milliliters of 50% (w/v) (NH4)2SO4, 60 milliliters of 10× trace metals solution, and 30 milliliters of 1 M MgSO4. Pichia methanolica fermentation is robust and requires high agitation, aeration, and oxygen sparging to maintain the percentage dissolved oxygen saturation above 30%. The percentage dissolved oxygen should not drop below 15% for optimal expression and growth. The biomass typically reaches about 30 to about 80 grams dry cell weight per liter at 48 hours EFT.
[0092]Proteins produced according to the present invention are recovered from the host cells using conventional methods. Secreted proteins are recovered from the conditioned culture medium using standard methods, also selected for the particular protein. See, in general, Scopes, Protein Purification: Principles and Practice, Springer-Verlag, New York, 1994.
[0093]The materials and methods of the present invention can be used to produce proteins of research, industrial, or pharmaceutical interest. Such proteins include enzymes, such as lipases, cellulases, and proteases; antibodies and fragments thereof; enzyme inhibitors, including protease inhibitors; growth factors such as platelet derived growth factor (PDGF), fibroblast growth factors (FGF), epidermal growth factor (EGF), vascular endothelial growth factors (VEGFs); glutamic acid decarboxylase (GAD); cytokines, such as erythropoietin, thrombopoietin, colony stimulating factors, interleukins, and interleukin antagonist; hormones, such as insulin, proinsulin, leptin, and glucagon; adipocyte complement related proteins, such as zsig37, zsig39, zacrp8 and the like; and receptors, including growth factor receptors, which can be expressed in truncated form ("soluble receptors") or as fusion proteins with, for example, immunoglobulin constant region sequences. DNAs encoding these and other proteins are known in the art. See, for example, U.S. Pat. Nos. 4,889,919; 5,219,759; 4,868,119; 4,968,607; 4,599,311; 4,784,950; 5,792,850; 5,827,734; 4,703,008; 4,431,740; 4,762,791; 6,265,544; 6,566,499; 6,197,930; 6,482,612; and WIPO Publications WO 95/21920 and WO 96/22308.
[0094]It is particularly preferred to use the present invention to produce unglycosylated pharmaceutical proteins. Yeast cells, including P. methanolica cells, produce glycoproteins with carbohydrate chains that differ from their mammalian counterparts. Mammalian glycoproteins produced in yeast cells may therefore be regarded as "foreign" when introduced into a mammal, and may exhibit, for example, different pharmacokinetics than their naturally glycosylated counterparts.
[0095]The present invention also provides antibodies to polypeptides of the present invention. Antibodies to β-glucanase can be obtained, for example, using as an antigen the product of β-glucanase expression vector or β-glucanase isolated from a natural source. Particularly useful anti-β-glucanase antibodies "bind specifically" with β-glucanase. Antibodies are considered to be specifically binding if the antibodies exhibit at least one of the following two properties: (1) antibodies bind to β-glucanase with a threshold level of binding activity, and (2) antibodies do not significantly cross-react with polypeptides related to β-glucanase.
[0096]With regard to the first characteristic, antibodies specifically bind if they bind to a β-glucanase polypeptide, peptide or epitope with a binding affinity (Ka) of 106M-1 or greater, preferably 107 M-1 or greater, more preferably 108 M-1 or greater, and most preferably 109 M-1 or greater. The binding affinity of an antibody can be readily determined by one of ordinary skill in the art, for example, by Scatchard analysis (Scatchard, Ann. NY Acad. Sci. 51:660 (1949)). With regard to the second characteristic, antibodies do not significantly cross-react with related polypeptide molecules, for example, if they detect β-glucanase, but not known related polypeptides using a standard Western blot analysis. Examples of known related polypeptides are orthologs and proteins from the same species that are members of a protein family.
[0097]Anti-β-glucanase antibodies can be produced using antigenic β-glucanase epitope-bearing peptides and polypeptides. Antigenic epitope-bearing peptides and polypeptides of the present invention contain a sequence of at least nine, at least 12, at least 15, at least 18, at least 21, or at least 24 to about 28 amino acids contained within SEQ ID NO:2. It is desirable that the amino acid sequence of the epitope-bearing peptide is selected to provide substantial solubility in aqueous solvents (i.e., the sequence includes relatively hydrophilic residues, while hydrophobic residues are preferably avoided). Moreover, amino acid sequences containing proline residues may be also be desirable for antibody production.
[0098]As an illustration, potential antigenic sites in β-glucanase can be identified using the Jameson-Wolf method, Jameson and Wolf, CABIOS 4:181, (1988), as implemented by the PROTEAN program (version 3.14) of LASERGENE (DNASTAR; Madison, Wis.). Default parameters were used in this analysis.
[0099]The Jameson-Wolf method predicts potential antigenic determinants by combining six major subroutines for protein structural prediction. Briefly, the Hopp-Woods method, Hopp et al., Proc. Nat'l Acad. Sci. USA 78:3824 (1981), is first used to identify amino acid sequences representing areas of greatest local hydrophilicity (parameter: seven residues averaged). In the second step, Emini's method, Emini et al, J. Virology 55:836 (1985), is used to calculate surface probabilities (parameter: surface decision threshold (0.6)=1). Third, the Karplus-Schultz method, Karplus and Schultz, Naturwissenschaften 72:212 (1985), is used to predict backbone chain flexibility (parameter: flexibility threshold (0.2)=1). In the fourth and fifth steps of the analysis, secondary structure predictions are applied to the data using the methods of Chou-Fasman, Chou, "Prediction of Protein Structural Classes from Amino Acid Composition," in Prediction of Protein Structure and the Principles of Protein Conformation, Fasman (ed.), pages 549-586 (Plenum Press 1990), and Garnier-Robson, Gamier et al, J. Mol. Biol. 120:97 (1978) (Chou-Fasman parameters: conformation table=64 proteins; α region threshold=103; β region threshold=105; Garnier-Robson parameters: α and β decision constants=0). In the sixth subroutine, flexibility parameters and hydropathy/solvent accessibility factors are combined to determine a surface contour value, designated as the "antigenic index." Finally, a peak broadening function is applied to the antigenic index, which broadens major surface peaks by adding 20%, 40%, 60%, or 80% of the respective peak value to account for additional free energy derived from the mobility of surface regions relative to interior regions. This calculation is not applied, however, to any major peak that resides in a helical region, since helical regions tend to be less flexible.
[0100]Polyclonal antibodies to recombinant β-glucanase protein or to β-glucanase isolated from natural sources can be prepared using methods well-known to those of skill in the art. Antibodies can also be generated using a β-glucanase-glutathione transferase fusion protein, which is similar to a method described by Burrus and McMahon, Exp. Cell Res. 220:363 (1995). General methods for producing polyclonal antibodies are described, for example, by Green et al., "Production of Polyclonal Antisera," in Immunochemical Protocols (Manson, ed.), pages 1-5 (Humana Press 1992), and Williams et al., "Expression of foreign proteins in E. coli using plasmid vectors and purification of specific polyclonal antibodies," in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al (eds.), page 15 (Oxford University Press 1995).
[0101]The immunogenicity of a β-glucanase polypeptide can be increased through the use of an adjuvant, such as alum (aluminum hydroxide) or Freund's complete or incomplete adjuvant. Polypeptides useful for immunization also include fusion polypeptides, such as fusions of β-glucanase or a portion thereof with an immunoglobulin polypeptide or with maltose binding protein. The polypeptide immunogen may be a full-length molecule or a portion thereof. If the polypeptide portion is "hapten-like," such portion may be advantageously joined or linked to a macromolecular carrier (such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid) for immunization.
[0102]Although polyclonal antibodies are typically raised in animals such as horse, cow, dog, chicken, rat, mouse, rabbit, goat, guinea pig, or sheep, an anti-β-glucanase antibody of the present invention may also be derived from a subhuman primate antibody. General techniques for raising diagnostically and therapeutically useful antibodies in baboons may be found, for example, in Goldenberg et al., International Patent Publication No. WO 91/11465, and in Losman et al., Int. J. Cancer 46:310 (1990).
[0103]Alternatively, monoclonal anti-β-glucanase antibodies, e.g., neutralizing monoclonal antibodies to neutralize β-glucanase activity, can be generated. Rodent monoclonal antibodies to specific antigens may be obtained by methods known to those skilled in the art (see, for example, Kohler et al., Nature 256:495 (1975), Coligan et al. (eds.), Current Protocols in Immunology, Vol. 1, pages 2.5.1-2.6.7 (John Wiley & Sons 1991) ["Coligan"], Picksley et al., "Production of monoclonal antibodies against proteins expressed in E. coli," in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al (eds.), page 93 (Oxford University Press 1995)).
[0104]Briefly, monoclonal antibodies can be obtained by injecting mice with a composition comprising a β-glucanase gene product, verifying the presence of antibody production by removing a serum sample, removing the spleen to obtain B-lymphocytes, fusing the B-lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, selecting positive clones which produce antibodies to the antigen, culturing the clones that produce antibodies to the antigen, and isolating the antibodies from the hybridoma cultures.
[0105]Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well-established techniques. Such isolation techniques include affinity chromatography with Protein-A Sepharose, size-exclusion chromatography, and ion-exchange chromatography (see, for example, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3; Baines et al., "Purification of Immunoglobulin G (IgG)," in Methods in Molecular Biology, Vol 10, pages 79-104 (The Humana Press, Inc. 1992)).
[0106]For particular uses, it may be desirable to prepare fragments of anti-β-glucanase antibodies. Such antibody fragments can be obtained, for example, by proteolytic hydrolysis of the antibody. Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. As an illustration, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab')2. This fragment can be further cleaved using a thiol reducing agent to produce 3.5S Fab' monovalent fragments. Optionally, the cleavage reaction can be performed using a blocking group for the sulfhydryl groups that result from cleavage of disulfide linkages. As an alternative, an enzymatic cleavage using pepsin produces two monovalent Fab fragments and an Fc fragment directly. These methods are described, for example, by Goldenberg, U.S. Pat. No. 4,331,647, Nisonoff et al., Arch Biochem. Biophys. 89:230 (1960), Porter, Biochem. J. 73:119 (1959), Edelman et al., in Methods in Enzymology Vol 1, page 422 (Academic Press 1967), and by Coligan at pages 2.8.1-2.8.10 and 2.10.-2.10.4.
[0107]Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.
[0108]For example, Fv fragments comprise an association of VH and VL chains. This association can be noncovalent, as described by Inbar et al., Proc. Nat'l Acad. Sci. USA 69:2659 (1972). Alternatively, the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde (see, for example, Sandhu, Crit. Rev. Biotech. 12:437 (1992)).
[0109]The Fv fragments may comprise VH and VL chains which are connected by a peptide linker. These single-chain antigen binding proteins (scFv) are prepared by constructing a structural gene comprising DNA sequences encoding the VH and VL domains which are connected by an oligonucleotide. The structural gene is inserted into an expression vector which is subsequently introduced into a host cell, such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing scFvs are described, for example, by Whitlow et al., Methods: A Companion to Methods in Enzymology 2:97 (1991) (also see, Bird et al., Science 242:423 (1988), Ladner et al., U.S. Pat. No. 4,946,778, Pack et al., Bio/Technology 11:1271 (1993), and Sandhu, supra).
[0110]As an illustration, a scFV can be obtained by exposing lymphocytes to β-glucanase polypeptide in vitro, and selecting antibody display libraries in phage or similar vectors (for instance, through use of immobilized or labeled β-glucanase protein or peptide). Genes encoding polypeptides having potential β-glucanase polypeptide binding domains can be obtained by screening random peptide libraries displayed on phage (phage display) or on bacteria, such as E. coli. Nucleotide sequences encoding the polypeptides can be obtained in a number of ways, such as through random mutagenesis and random polynucleotide synthesis. These random peptide display libraries can be used to screen for peptides which interact with a known target which can be a protein or polypeptide, such as a ligand or receptor, a biological or synthetic macromolecule, or organic or inorganic substances. Techniques for creating and screening such random peptide display libraries are known in the art (Ladner et al., U.S. Pat. No. 5,223,409, Ladner et al., U.S. Pat. No. 4,946,778, Ladner et al., U.S. Pat. No. 5,403,484, Ladner et al., U.S. Pat. No. 5,571,698, and Kay et al., Phage Display of Peptides and Proteins (Academic Press, Inc. 1996)) and random peptide display libraries and kits for screening such libraries are available commercially, for instance from CLONTECH Laboratories, Inc. (Palo Alto, Calif.), Invitrogen Inc. (San Diego, Calif.), New England Biolabs, Inc. (Beverly, Mass.), and Pharmacia LKB Biotechnology Inc. (Piscataway, N.J.). Random peptide display libraries can be screened using the β-glucanase sequences disclosed herein to identify proteins which bind to β-glucanase.
[0111]Another form of an antibody fragment is a peptide coding for a single complementarity-determining region (CDR). CDR peptides ("minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells (see, for example, Larrick et al., Methods: A Companion to Methods in Enzymology 2:106 (1991), Courtenay-Luck, "Genetic Manipulation of Monoclonal Antibodies," in Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al. (eds.), page 166 (Cambridge University Press 1995), and Ward et al., "Genetic Manipulation and Expression of Antibodies," in Monoclonal Antibodies: Principles and Applications, Birch et al., (eds.), page 137 (Wiley-Liss, Inc. 1995)).
[0112]Polyclonal anti-idiotype antibodies can be prepared by immunizing animals with anti-β-glucanase antibodies or antibody fragments, using standard techniques. See, for example, Green et al., "Production of Polyclonal Antisera," in Methods In Molecular Biology: Immunochemical Protocols, Manson (ed.), pages 1-12 (Humana Press 1992). Also, see Coligan at pages 2.4.1-2.4.7. Alternatively, monoclonal anti-idiotype antibodies can be prepared using anti-β-glucanase antibodies or antibody fragments as immunogens with the techniques, described above.
[0113]Anti-idiotype β-glucanase antibodies, as well as β-glucanase polypeptides, can be used to identify and to isolate β-glucanase substrates and inhibitors. For example, proteins and peptides of the present invention can be immobilized on a column and used to bind substrate and inhibitor proteins from biological samples that are run over the column (Hermanson et al. (eds.), Immobilized Affinity Ligand Techniques, pages 195-202 (Academic Press 1992)). Radiolabeled or affinity labeled β-glucanase polypeptides can also be used to identify or to localize β-glucanase substrates and inhibitors in a biological sample (see, for example, Deutscher (ed.), Methods in Enzymol., vol. 182, pages 721-37 (Academic Press 1990); Brunner et al., Ann. Rev. Biochem. 62:483 (1993); Fedan et al., Biochem. Pharmacol 33:1167 (1984)).
[0114]The present invention also provides DNA molecules, such as DNA constructs containing a functional β-glucanase gene, in a kit. Alternatively, such a kit may include Pichia methanolica cells, such as deficient in AUG1 and/or AUG2 promoter and vacuolar proteinase A and/or vacuolar proteinase B. Moreover, the kit may include instructions on how to insert a gene encoding a protein of interest into the DNA construct as well as instructions on how to transform the provided Pichia methanolica cells, and express, produce and recover the protein of interest.
[0115]The invention is further illustrated by the following nonlimiting examples.
EXAMPLES
Example 1
Identification of exo-1,3-β-glucanase
[0116]To clone the P. methanolica β-glucanase gene, a 45 kDa secreted protein was isolated from PMAD16 strain broth grown under fermentation conditions. N-terminal sequencing verified that the protein isolated was found to have 76.7% homology to the corresponding H. polymorpha exo-1,3-β-glucanase protein sequence and a 74.1% homology to the corresponding S. occidentalis exo-1,3-β-glucanase protein sequence within a 30 amino acid overlap. Degenerate sense (ZC18,176; SEQ ID NO:7 and ZC18,177; SEQ ID NO:8) and antisense (ZC16,562; SEQ ID NO:9 and ZC16,567; SEQ ID NO:10 and ZC18,180; SEQ ID NO:11 and ZC18,181; SEQ ID NO:12) PCR primers were designed from an alignment of the coding regions of the exo-1,3-β-glucanase genes of H. polymorpha and S. occidentalis. The primers were then used to amplify P. methanolica genomic DNA. An amplified sequence 1280 bp long was recovered and found to have 65.0% homology to the corresponding H. polymorpha exo-1,3-β-glucanase protein sequence.
[0117]A P. methanolica genomic library was constructed in the vector pRS426 (Christianson et al., Gene 110:119-122, 1992), a shuttle vector comprising 2μ and S. cerevisiae URA3 sequences, allowing it to be propagated in S. cerevisiae. Genomic DNA was prepared from strain CBS6515 according to standard procedures. Briefly, cells were cultured overnight in rich media, spheroplasted with zymolyase, and lysed with SDS. DNA was precipitated from the lysate with ethanol and extracted with a phenol/chloroform mixture, then precipitated with ammonium acetate and ethanol. Gel electrophoresis of the DNA preparation showed the presence of intact, high molecular weight DNA and appreciable quantities of RNA. The DNA was partially digested with Sau 3A by incubating the DNA in the presence of a dilution series of the enzyme. Samples of the digests were analyzed by electrophoresis to determine the size distribution of fragments. DNA migrating between 4 and 12 kb was cut from the gel and extracted from the gel slice. The size-fractionated DNA was then ligated to pRS426 that had been digested with Bam HI and treated with alkaline phosphatase. Aliquots of the reaction mixture were electroporated into E. coli MC1061 cells using an electroporator (Gene Pulser®; BioRad Laboratories, Hercules, Calif.) as recommended by the manufacturer.
[0118]The library was screened by PCR using sense and antisense primers designed from the sequenced region of the P. methanolica exo-1,3-β-glucanase gene fragment. The PCR reaction mixture was incubated for one minute at 94° C.: followed by 34 cycles of 94° C., one minute, 52° C., one minute, 72° C., eleven minutes. Starting with 43 library pools, positive pools were identified and broken down to individual colonies. A single colony with a pRS426 plasmid containing the P. methanolica exo-1,3-β-glucanase gene as its insert was isolated. The orientation of the exo-1,3-glucanase gene and the length of the 5' and 3' flanking sequences in the insert were deduced by DNA sequencing (SEQ ID NO:1). This gene was designated exo-1,3-β-glucanase.
Example 2
Construction and Characterization of ZACRP3 Untagged Yeast Expression Vectors Utilizing a Heterologous S. cerevisiae Leader and an Endogenous P. methanolica Leader
[0119]Expression of zacrp3 (Piddington et al., U.S. Pat. No. 6,521,233) in Pichia methanolica utilizes the expression system as described in Raymond, U.S. Pat. No. 5,888,768; Raymond, U.S. Pat. No. 5,955,349; and Raymond, U.S. Pat. No. 6,001,597. An expression plasmid containing all or part of a polynucleotide encoding zacrp3 is constructed via homologous recombination (Raymond et al., U.S. Pat. No. 5,854,039). An expression vector was built from pVRM51 to express untagged zacrp3 polypeptides. PVRM51 is a derivative of the pCZR204 expression vector; it differs from pCZR204 by one amino acid (D83->Y83) within the alpha factor prepro (αFpp) sequence to enhance Kex2p cleavage. The pVRM51 vector contains the AUG1 promoter, followed by the αFpp (D83->Y83) leader sequence and an amino-terminal peptide tag (Glu-Glu), followed by a blunt-ended Sma I restriction site, a carboxy-terminal peptide tag (Glu-Glu), a translational STOP codon, followed by the AUG1 terminator, the ADE2 selectable marker, and finally the AUG1 3' untranslated region. Also included in this vector are the URA3 and CEN-ARS sequences required for selection and replication in S. cerevisiae, and the AmpR and colE1 ori sequences required for selection and replication in E. coli. A second expression vector was built from zCZR204 to express untagged zacrp3 polypeptides. The zCZR204 expression vector is as described above, the only difference is that this expression plasmid has the β-glucanase leader inserted where the αFpp leader usually is. The zacrp3 sequence inserted into these vectors begins at residue 23 (Gln) of the zacrp3 amino acid sequence. The nucleotide sequence of zacrp3 is shown in SEQ ID NO:13 and the polypeptide sequence of zacrp3 is shown in SEQ ID NO:14.
[0120]For each construct specific recombination primers were designed. For the αFppD->Y::zacrp3 construct, these primers are ZG37,475 (SEQ ID NO:15) and ZG37,474 (SEQ ID NO:16). For the β-glucanase::zacrp3 construct, the β-glucanase leader was amplified using primers ZG39,207 (SEQ ID NO:17) and ZG39,209 (SEQ ID NO:18), while zacrp3 was amplified using primers ZG39,208 (SEQ ID NO:19) and ZG37,474 (SEQ ID NO:16). The resulting PCR fragments were homologously recombined into the yeast expression vectors described above. For the αFppD->Y::zacrp3 construct, the N-terminal primer (ZG37,475) (SEQ ID NO: 15) spans 39 base pairs of the alpha factor prepro (αFpp) coding sequence on one end, followed by 26 base pairs of the amino-terminus coding sequence of mature zacrp3 sequence on the other. The C-terminal primer (ZG37,474) (SEQ ID NO:16) spans about 28 base pairs of carboxy terminus coding sequence of zacrp3 on one end with 40 base pairs of AUG1 terminator sequence.
[0121]For the β-glucanase::zacrp3 construct, the N-terminal β-glucanase primer (ZG39,207) (SEQ ID NO:17) spans 40 base pairs of AUG1p sequence, followed by 27 base pairs of β-glucanase leader sequence. The C-terminal primer (ZG39,209) (SEQ ID NO: 18) that amplifies β-glucanase contains 30 base pairs of carboxy terminus coding sequence of β-glucanase followed by 33 base pairs of the amino-terminus coding sequence of the Glu-Glu tag. The N-terminal zacrp3 primer (ZG39,208) (SEQ ID NO:19) spans 39 base pairs of β-glucanase sequence, followed by 26 base pairs of the mature zacrp3 sequence. The C-terminal primer (ZG37,474) (SEQ ID NO:16) that amplifies zacrp3 spans about 28 base pairs of carboxy terminus coding sequence of zacrp3 on one end with 40 base pairs of AUG1 terminator sequence.
Construction of the Untagged zacrp3Plasmid Utilizing the αFpp Leader
[0122]An untagged zacrp3 plasmid was made by homologously recombining 100 ng of the SmaI digested pVRM51 acceptor vector and 1 μg of PCR amplified zacrp3 cDNA donor fragment, in S. cerevisiae SF838-9Dα.
[0123]The zacrp3 PCR fragment was synthesized by a PCR reaction. To a final reaction volume of 100 μl was added 100 pmol each of primers, ZG37,474 (SEQ ID NO:16) and ZG37,475 (SEQ ID NO:15), 10 μl of 10× PCR buffer (Boehringer Mannheim), 1 μL Pwo Polymerase (Boehringer Mannheim), 10 μL of 0.25 mM nucleotide triphosphate mix (Perkin Elmer) and dH2O. The PCR reaction was run 1 cycle at 2 minutes at 94° C., followed by 25 cycles of 30 seconds at 94° C., 1 minute at 50° C. and 1 minute at 72° C., followed by a 7 minute extension at 72° C., and concluded with an overnight hold at 4° C. The resulting 754 bp double stranded, zacrp3 fragment is disclosed in SEQ ID NO:20.
Construction of the Untagged zacrp3 Plasmid Utilizing the β-glucanase Leader
[0124]An untagged zacrp3 plasmid was made by homologously recombining 100 ng of the SmaI digested pCZR204 acceptor vector and 1 μg each of PCR amplified β-glucanase leader donor fragment and 1 μg zacrp3 cDNA donor fragment, in S. cerevisiae SF838-9Dα. The zacrp3 PCR fragments were synthesized by first amplifying the two fragments containing the β-glucanase leader and zacrp3, respectively, in separate reactions.
[0125]The β-glucanase leader was amplified in a PCR reaction as follows: to a final reaction volume of 100 μl was added 100 pmol each of primers, ZG39,207 (SEQ ID NO:17) and ZG39,209 (SEQ ID NO:18), 10 μl of 10× PCR buffer (Boehringer Mannheim), 1 μL Pwo Polymerase (Boehringer Mannheim), 10 μL of 0.25 mM nucleotide triphosphate mix (Perkin Elmer) and dH2O. The PCR reaction was run 1 cycle at 2 minutes at 94° C., followed by 25 cycles of 30 seconds at 94° C., 1 minute at 50° C. and 30 seconds at 72° C., followed by a 7 minute extension at 72° C., and concluded with an overnight hold at 4° C. The resulting 157 bp double stranded, β-glucanase leader fragment is disclosed in SEQ ID NO:21.
[0126]Zacrp3 was amplified in an additional PCR reaction as follows: to a final reaction volume of 100 μl was added 100 pmol each of primers, ZG39,208 (SEQ ID NO:19) and ZG37,474 (SEQ ID NO:16), 10 μL of 10× PCR buffer (Boehringer Mannheim), 1 μL Pwo Polymerase (Boehringer Mannheim), 10 μL of 0.25 mM nucleotide triphosphate mix (Perkin Elmer) and dH2O. The PCR reaction was run 1 cycle at 2 minutes at 94° C., followed by 25 cycles of 30 seconds at 94° C., 1 minute at 50° C. and 30 seconds at 72° C., followed by a 7 minute extension at 72° C., and concluded with an overnight hold at 4° C. The resulting 754 bp fragment is double stranded, and the zacrp3 PCR fragment is disclosed in SEQ ID NO:22.
[0127]One hundred microliters of competent yeast cells (S. cerevisiae strain SF838-9Dα) was independently combined with the various DNA mixtures from above and transferred to a 0.2 cm electroporation cuvette. The yeast/DNA mixtures were electropulsed at 0.75 kV (5 kV/cm), infinite Ω, 25 μF. The yeast/DNA mixtures were then added to 1 ml of 1.2 M sorbitol and incubated at 30° C. for 1 hour. The yeast was then plated in two 500 μl aliquots onto two URA DS plates and incubated at 30° C.
[0128]After about 48 hours the Ura.sup.+ yeast transformants from a single plate were resuspended in 1 ml H2O and spun briefly to pellet the yeast cells. The cell pellet was resuspended in 300 μL of Qiagen P1 lysis buffer and transferred to a fresh tube that contained 100-200 μL acid-washed glass beads (Sigma). Samples were vortexed for 1 minute intervals two or three times to lyse cells. Samples were allowed to settle, and 250 μl lysate was transferred to a fresh tube and the remainder of the Qiagen Spin Miniprep Kit was carried out following manufacterer's instructions.
[0129]Transformation of electrocompetent E. coli DH10B cells (Invitrogen) was done with 2 μl yeast DNA prep and 40 ul of DH10B cells. The cells were electropulsed in 0.1 cm cuvettes at 2.0 kV, 25 μF and 100 Ω. Following electroporation, 250 μl SOC (2% Bacto Tryptone (Difco, Detroit, Mich.), 0.5% yeast extract (Difco), 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM glucose) was plated in one aliquot on an LB AMP plate (LB broth (Lennox), 1.8% Bacto Agar (Difco), 100 mg/L Ampicillin). Plates were incubated at 37° C. overnight.
[0130]Individual clones harboring the correct expression construct for untagged zacrp3 were identified by restriction digest to verify the presence of the zacrp3 insert and to confirm that the various DNA sequences had been joined correctly to one another. The inserts of positive clones were subjected to sequence analysis. The αFpp D->Y leader::zacrp3 plasmid was designated pSDH147 and the β-glucanase leader::zacrp3 plasmid was designated pSDH149. Larger scale plasmid DNA was isolated for both plasmids using the Qiagen Maxi kit (Qiagen) according to manufacturer's instruction and the DNA was digested with Not I to liberate the Pichia-zacrp3 expression cassette from the vector backbone. The Not I-restriction digested DNA fragment was then transformed into the Pichia methanolica expression hosts, PMAD16 and PMAD18. This was done by mixing 100 μl of prepared competent PMAD16 or PMAD18 cells with 10 μg of Not I restriction digested pSDH147 or pSDH149, in separate transformations, and transferred to a 0.2 cm electroporation cuvette. The yeast/DNA mixture was electropulsed at 0.75 kV, 25 μF, infinite Ω. To the cuvette was added 800 μl of 1.2M Sorbitol and 400 μl aliquots were plated onto two ADE DS (0.056% -Ade -Trp -Thr powder, 0.67% yeast nitrogen base without amino acids, 2% D-glucose, 0.5% 200× tryptophan, threonine solution, and 18.22% D-sorbitol) plates for selection and incubated at 30° C.
Zacrp3 Expression in P. methanolica hosts PMAD16 and PMAD18-Clone Selection and Characterization
[0131]One hundred clones of each strain/plasmid (for 400 clones total) were isolated. Of these, only 10 of each were screened via Western blot for high-level zacrp3 expression. All 40 clones were grown in the following manner: 25 ml cultures of each were inoculated using one colony of each strain in BMY.1 pH6.0 media (Per liter: 13.4 g Yeast Nitrogen Base without amino acids (Becton Dickinson), 10.0 g Yeast Extract (Difco), 10.0 g tryptone (Difco), 10.0 g casamino acids (Difco), 6.7 g K2HPO4 (EM Science), 4.2 g citric acid (EM Science), and water) +2% glucose. BMY.1 media was supplemented with 10 mls per liter of media with FXIII vitamin solution (0.05 g/L biotin, 0.8 g/L thiamine hydrochloride, 0.8 g/L pyroxidine HCL, 15.0 g/L inositol, 15.0 g/L calcium pantothenate, 0.6 g/L niacinamide, 0.1 g/L folic acid, 0.2 g/L riboflavin, 1.0 g/L choline chloride). Cultures were grown in 125 ml baffled flasks on a platform shaker set to 250 rpm at 30° C. overnight.
[0132]The following day, 1 ml of each overnight inoculum culture was diluted into 24 mls of fresh BMY.1 media supplemented with FXIII vitamins as above, +1% Methanol to induce the AUG1 promoter (no glucose was added). Cultures were grown in 125 ml baffled flasks on a platform shaker set to 250 rpm at 30° C. for 24 hours. After 24 hours of growth and induction, the cultures were harvested at 500 rpm for 10 minutes in a Beckman centrifuge (JA-20 rotor) to pellet the cells. Three hundred μL of zacrp3 containing supernatant was mixed with 100 μL of NuPAGE 4× Sample Buffer (Invitrogen). Each 400 μl sample was split into two 200 μl samples: one set of samples was treated with 2% β-mercaptoethanol (Sigma) and represents a reduced sample, while the other set represents the non-reduced sample.
[0133]An SDS-PAGE analysis was carried out as described below. All reduced samples were heated for 10 min at 100° C., while all non-reduced samples were heated for 10 min at 65° C. Fifteen μL of each sample was applied for electrophoresis on a polyacrylamide gel. Protein separation was performed by electrophoresis in a 4-12% gradient NuPAGE polyacrylamide resolving gel (Invitrogen) under denaturing conditions (SDS-PAGE) using 1× MES running buffer (Invitrogen). The voltage of 130 V was applied throughout the entire run. Subsequently, electrotransference was carried out to a 0.2 μn nitrocellulose membrane (Invitrogen) for 1 h at 400 mA (constant current). The blots were then incubated for 30 minutes with agitation at 40 rpm in a blocking solution [Western A+10% non-fat dry milk (NFDM)(Carnation)] in order to block the protein-free areas of the membrane at 25° C.
[0134]As the first antibody, an anti-zacrp3 affinity purified antibody, E1834, developed in the rabbit (in-house) was used in a dilution of 1:10,000 in Western A +2.5% NFDM. Incubation was 2 hours at 25° C. Subsequently two 5 min. washings were performed at moderate agitation with Western B, followed by one 5 minute was at moderate agitation with Western A. As the second antibody a rabbit anti-IgG developed in the goat (Amersham) was used in a dilution of 1:2000 in Western A +2.5% NFDM. Blots were incubated for 1 hour at room temperature and washed three times for 5 min with moderate agitation with Western B, followed by a brief rinse in dH2O. Two mls of both Enhanced Chemiluminescent substrates (Amersham) were mixed together at a 1:1 ratio, and the blots were incubated in this solution for 5 seconds prior to development. The exposed blots were then developed using timed exposure to X-ray film (Kodak) and the film was subsequently developed to visualize data.
[0135]The electrophoretic analysis on the polyacrylamide gel of the culture medium from P. methanolica clones representing pSDH149 (β-glucanase leader) and pSDH147 (S. cerevisiae alpha factor pre-pro sequence) showed that in the culture medium from both host strains a band of approximately 28 kDa (under reduced conditions) appears corresponding to zacrp3, while in the non-induced cell culture medium, there was no band. Roughly ninety percent of the recombinant clones that were analyzed for the integrated heterologous gene expression produced and secreted recombinant zacrp3. The resulting zacrp3 plasmid-containing yeast strains show the endogenous P. methanolica β-glucanase leader construct pSDH149 secretes equivalent levels of zacrp3 compared to the heterologous S. cerevisiae αFpp leader pSDH147 in the PMAD16 host strain background. Interestingly, plasmid-containing yeast strains show the endogenous P. methanolica β-glucanase leader construct pSDH149 secretes approximately 2-3 fold higher levels of zacrp3 compared to the heterologous S. cerevisiae αFpp leader pSDH147 in the PMAD18 host strain background. One isolet of each αFpp::zacrp3 strain was picked for subsequent use; the resulting clones were designated PMAD16::pSDH147.4.2, PMAD18::pSDH147.4.8, respectively. Two isolets of each β-glucanase::zacrp3 strain was picked for subsequent use; the resulting clones were designated PMAD16::pSDH149.4.4, PMAD16::pSDH149.4.9, PMAD 18::pSDH149.4.5, and PMAD 18: :pSDH149.4.8, respectively.
Example 3
Construction and Characterization of Zsig3 7 Untagged Yeast Expression Vectors Utilizing a Heterologous S. cerevisiae Leader and an Endogenous P. methanolica Leader
[0136]Expression of zsig37 in Pichia methanolica utilizes the expression system as described in Raymond, U.S. Pat. No. 5,888,768; Raymond, U.S. Pat. No. 5,955,349; and Raymond, U.S. Pat. No. 6,001,597. An expression plasmid containing all or part of a polynucleotide encoding zsig37 is constructed via homologous recombination (Raymond et al., U.S. Pat. No. 5,854,039). Zsig37 was recombined into the vector pCZR204. Oligos used to amplify zsig37 introduced a single amino acid mutation (D83->Y83) within the alpha factor prepro (αFpp) sequence to enhance Kex2p cleavage. This mutation was then introduced into the vector pCZR204 when recombination occurred. The pCZR204 vector contains the AUG1 promoter, followed by the αFpp leader sequence and an amino-terminal peptide tag (Glu-Glu), followed by a blunt-ended Sma I restriction site, a carboxy-terminal peptide tag (Glu-Glu), a translational STOP codon, followed by the AUG1 terminator, the ADE2 selectable marker, and finally the AUG1 3' untranslated region. Also included in this vector are the URA3 and CEN-ARS sequences required for selection and replication in S. cerevisiae, and the AmpR and colE1 ori sequences required for selection and replication in E. coli. A second expression vector was built from zCZR204 to express untagged zsig37 polypeptides. The zCZR204 expression vector is as described above, the only difference is that this expression plasmid has the β-glucanase leader inserted where the αFpp leader usually is. The zsig37 sequence inserted into these vectors begins at residue 86 (Arg) of the zsig37 amino acid sequence. The full-length nucleotide sequence of zsig37 is shown in SEQ ID NO:27 and the full-length polypeptide sequence of zsig37 is shown in SEQ ID NO:28 (See U.S. Pat. Nos. 6,265,544, 6,566,499, 6,518,403, 6,448,221, and 6,544,946).
[0137]For each construct specific recombination primers were designed. For the αFppD83->Y83::zsig37 construct, these primers are ZG42,210 (SEQ ID NO:29) and ZG42,206 (SEQ ID NO:30). For the β-glucanase::zsig37 construct, the β-glucanase leader was amplified using primers ZG42,209 (SEQ ID NO:31) and ZG42,211 (SEQ ID NO:32), while zsig37 was amplified using primers ZG42,273 (SEQ ID NO:33) and ZG42,206 (SEQ ID NO:30). The resulting PCR fragments were homologously recombined into the yeast expression vector described above. For the αFppD83->Y83::zsig37 construct, the N-terminal primer (ZG42,210) (SEQ ID NO:29) spans 39 base pairs of the alpha factor prepro (αFpp) coding sequence on one end, and introduces the D83->Y83 mutation in the quadratureFFpp sequence, followed by 25 base pairs of the amino-terminus coding sequence of mature zsig37 sequence on the other. The C-terminal primer (ZG42,206) (SEQ ID NO:30) spans about 21 base pairs of carboxy terminus coding sequence of zsig37 on one end with 40 base pairs of AUG1 terminator sequence.
[0138]For the β-glucanase::zsig37 construct, the N-terminal β-glucanase primer (ZG42,209) (SEQ ID NO:3 1) spans 40 base pairs of AUG1p sequence, followed by 27 base pairs of β-glucanase leader sequence. The C-terminal primer (ZG42,211) (SEQ ID NO:32) that amplifies β-glucanase contains 39 base pairs of carboxy terminus coding sequence of β-glucanase followed by 25 base pairs of the amino-terminus coding sequence of the mature zsig37 sequence. The N-terminal zsig37 primer (ZG42,273) (SEQ ID NO:33) spans 39 base pairs of β-glucanase sequence, followed by 25 base pairs of the mature zsig37 sequence. The C-terminal primer (ZG42,206) (SEQ ID NO:30) that amplifies zsig37 spans about 21 base pairs of carboxy terminus coding sequence of zsig37 on one end with 40 base pairs of AUG1 terminator sequence.
Construction of the Untagged zsig37 Plasmid Utilizing the αFppD->Y Leader
[0139]An untagged zsig37 plasmid was made by homologously recombining 100 ng of the SmaI digested pCZR204 acceptor vector and 1 μg of PCR amplified zsig37 cDNA donor fragment, in S. cerevisiae SF838-9Dα.
[0140]The zsig37 PCR fragment was synthesized by a PCR reaction. To a final reaction volume of 100 μl was added 100 pmol each of primers, ZG42,210 (SEQ ID NO:29) and ZG42,206 (SEQ ID NO:30), 10 μl of 10× PCR buffer (Boehringer Mannheim), 1 μl Pwo Polymerase (Boehringer Mannheim), 10 μl of 0.25 mM nucleotide triphosphate mix (Perkin Elmer) and dH2O. The PCR reaction was run 1 cycle at 2 minutes at 94° C., followed by 30 cycles of 30 seconds at 94° C., 1 minute at 50° C. and 1 minute at 72° C., followed by a 7 minute extension at 72° C., and concluded with an overnight hold at 4° C. The resulting 846 bp double stranded, zsig37 fragment is disclosed in SEQ ID NO:34. The αFpp:zsig37 full-length nucleotide (pSDH156) is shown in SEQ ID NO:35, with its corresponding encoded protein shown in SEQ ID NO:36.
Construction of the Untagged zsig37 Plasmid Utilizing the β-glucanase Leader
[0141]An untagged zsig37 plasmid was made by homologously recombining 100 ng of the SmaI digested pCZR204 acceptor vector and 1 μg each of PCR amplified β-glucanase leader donor fragment and 1 μg zsig37 cDNA donor fragment, in S. cerevisiae SF838-9Dα. The zsig37 PCR fragments were synthesized by first amplifying the two fragments containing the β-glucanase leader and zsig37, respectively, in separate reactions.
[0142]The β-glucanase leader was amplified in a PCR reaction as follows: to a final reaction volume of 100 μl was added 100 pmol each of primers, ZG42,209 (SEQ ID NO:31) and ZG42,211 (SEQ ID NO:32), 10 μl of 10× PCR buffer (Boehringer Mannheim), 1 μl Pwo Polymerase (Boehringer Mannheim), 10 μl of 0.25 mM nucleotide triphosphate mix (Perkin Elmer) and dH2O. The PCR reaction was run 1 cycle at 2 minutes at 94° C., followed by 30 cycles of 30 seconds at 94° C., 1 minute at 50° C. and 1 minute at 72° C., followed by a 7 minute extension at 72° C., and concluded with an overnight hold at 4° C. The resulting 148 bp double stranded, β-glucanase leader fragment is disclosed in SEQ ID NO:37.
[0143]Zsig37 was amplified in an additional PCR reaction as follows: to a final reaction volume of 100 μl was added 100 pmol each of primers, ZG42,273 (SEQ ID NO:33) and ZG42,206 (SEQ ID NO:30), 10 μl of 10× PCR buffer (Boehringer Mannheim), 1 μl Pwo Polymerase (Boehringer Mannheim), 10 μl of 0.25 mM nucleotide triphosphate mix (Perkin Elmer) and dH2O. The PCR reaction was run 1 cycle at 2 minutes at 94° C., followed by 30 cycles of 30 seconds at 94° C., 1 minute at 50° C. and 1 minute at 72° C., followed by a 7 minute extension at 72° C., and concluded with an overnight hold at 4° C. The resulting 846 bp fragment is double stranded, and the zsig37 PCR fragment is disclosed in SEQ ID NO:38.
[0144]One hundred microliters of competent yeast cells (S. cerevisiae strain SF838-9Dα) was independently combined with the various DNA mixtures from above and transferred to a 0.2 cm electroporation cuvette. The yeast/DNA mixtures were electropulsed at 0.75 kV (5 kV/cm), infinite Ω, 25 μF. The yeast/DNA mixtures were then added to 1 ml of 1.2 M sorbitol and incubated at 30° C. for 1 hour. The yeast was then plated in two 500 μl aliquots onto two URA DS plates and incubated at 30° C.
[0145]After about 48 hours the Ura.sup.+ yeast transformants from a single plate were resuspended in 1 ml H2O and spun briefly to pellet the yeast cells. The cell pellet was resuspended in 300 μl of Qiagen P1 lysis buffer and transferred to a fresh tube that contained 100-200 μl acid-washed glass beads (Sigma). Samples were vortexed for 1 minute intervals two or three times to lyse cells. Samples were allowed to settle, and 250 μl lysate was transferred to a fresh tube and the remainder of the Qiagen Spin Miniprep Kit was carried out following manufacterer's instructions.
[0146]Transformation of electrocompetent E. coli DH10B cells (Invitrogen) was done with 2 μl yeast DNA prep and 40 ul of DH10B cells. The cells were electropulsed in 0.1 cm cuvettes at 2.0 kV, 25 μF and 100 Ω. Following electroporation, 250 μl SOC (2% Bacto Tryptone (Difco, Detroit, Mich.), 0.5% yeast extract (Difco), 10 mM NaCl (J. T. Baker), 2.5 mM KCl (Mallinkrodt), 10 mM MgCl2 (Mallinkrodt), 10 mM MgSO4 (J. T. Baker), 20 mM glucose (Difco) and water) was plated in one aliquot on an LB AMP plate (LB broth (Lennox), 1.8% Bacto Agar (Difco), 100 mg/L Ampicillin (Sigma)). Plates were incubated at 37° C. overnight.
[0147]Individual clones harboring the correct expression construct for untagged zsig37 were identified by restriction digest to verify the presence of the zsig37 insert and to confirm that the various DNA sequences had been joined correctly to one another. The inserts of positive clones were subjected to sequence analysis. The αFpp D83->Y83 leader::zsig37 plasmid was designated pSDH156 and the β-glucanase leader::zsig37 plasmid was designated pSDH160. Larger scale plasmid DNA was isolated for both plasmids using the Qiagen Maxi kit (Qiagen) according to manufacturer's instruction and the DNA was digested with Not I to liberate the Pichia-zsig37 expression cassette from the vector backbone. The Not I-restriction digested DNA fragment was then transformed into the Pichia methanolica expression hosts, PMAD16 and PMAD18. This was done by mixing 100 μl of prepared competent PMAD16 or PMAD18 cells with 1.0 μg and 2.5quadratureg of Not I restriction digested pSDH156 or pSDH160, in separate transformations, and transferred to a 0.2 cm electroporation cuvette. The yeast/DNA mixture was electropulsed at 0.75 kV, 25 μF, infinite Ω. To the cuvette was added 800 μl of 1.2M Sorbitol. Transformants were outgrown in test tubes at 30° C. for 2 hours prior to plating on selection plates. Four hundred μl aliquots were plated onto two ADE DS (0.056% -Ade -Trp -Thr powder (TCI America, Alfa Aesar, and Calbiochem), 0.67% yeast nitrogen base without amino acids (Becton Dickinson), 2% D-glucose (Difco), 0.5% 200× tryptophan, threonine solution (ICN and Alfa Aesar), and 18.22% D-sorbitol) plates for selection and incubated at 30° C. The β-glucanase::zsig37 full-length nucleotide sequence (pSDH160) is shown in SEQ ID NO:39, with its corresponding encoded protein shown in SEQ ID NO:40.
Zsig37 Expression in P. methanolica hosts PMAD16 and PMAD18-Clone Selection and Characterization
[0148]Two hundred fifty clones of PMAD16::pSDH156 and 300 clones of PMAD18::pSDH156 were isolated. In addition, 55 clones of PMAD16::pSDH160 and 68 clones of PMAD18::pSDH160 were isolated. All clones were screened via colony blot analysis for high-level zsig37 expression. Clones were screened by colony blot as follows: each transformant was patched to two fresh 1% Methanol plates (Per liter: 6.8 g Yeast Nitrogen Base without amino acids (Becton Dickinson), 0.6 g -ade -trp -thr powder (TCI America, Alfa Aesar, Calbiochem), 18.0 g Bacto agar (Difco), 5 mls 200× Tryptophan/threonine solution (Alfa Aesar and ICN), 10 mls Methanol (J. T. Baker), 2 mls saturated biotin (ICN) and water). Each plate was overlayed with a nitrocellulose filter (Schleicher & Schuell) and incubated at 30° C. for 3 days. Nitrocellulose filters were then removed. One set of filters was denatured and reduced under the following conditions: filters were placed in a hybridization tube and 25 mls of 25 mM Tris (Millipore), 25 mM Glycine (J. T. Baker), 5 mM quadrature-ME (Sigma) pH9.0 was added to each tube. Filters were incubated at 65° C. for 10 minutes. Post-denaturation/reduction, filters were removed and placed directly in Western block solution (50 mM Tris (Millipore) pH7.4, 5 mM EDTA (J. T. Baker) pH8.0, 0.05% Igepal CA-630 (Sigma), 150 mM NaCl (J. T. Baker), 2.5% Gelatin (Mallinkrodt), water and 10% nonfat dry milk (NFDM)(Carnation)). The other identical set of filters represents a non-denatured, non-reduced set of filters. These filters were removed from the plates and placed directly into Western block solution. All filters were incubated in block solution for 30 minutes at 25° C.
[0149]Filters were then incubated in Western A (50 mM Tris (Millipore) pH7.4, 5 mM EDTA (J. T. Baker) pH8.0, 0.05% Igepal CA-630 (Sigma), 150 mM NaCl (J. T. Baker), 2.5% Gelatin (Mallinkrodt), water) +2.5% NFDM (Carnation) containing 0.2 quadratureg/ml zsig37 primary antibody E1489 for 1-2 hours at 25° C. Blots were then washed 3 times for 7 minutes each at 25° C. in Western B (1M NaCl (J. T. Baker), 50 mM Tris (Millipore) pH7.4, 5 mM EDTA (J. T. Baker), 0.05% Igepal (Sigma), 0.25% gelatin (Mallinkrodt), and water) followed by one wash in Western A for 7 minutes at 25° C. Filters were then incubated in Western A +2.5% NFDM containing a 1:5000 dilution of donkey anti rabbit secondary antibody (Life Technologies) for 1 hour at 25° C. Blots were then washed 4 times for 7 minutes each at 25° C. in Western B (1M NaCl (J. T. Baker), 50 mM Tris (Millipore) pH7.4, 5 mM EDTA (J. T. Baker), 0.05% Igepal (Sigma), 0.25% gelatin (Mallinkrodt), and water) at 25° C. All blots were then briefly rinsed with deionized water before being developed with Lumi-Light Plus ECL substrate (Roche). Two mls of both Lumi-Light substrates were mixed together at a 1:1 ratio, and the blots were incubated in this solution for 5 seconds prior to development. The exposed blots were then developed using timed exposure to X-ray film (Kodak) and the film was subsequently developed to visualize data.
[0150]Ten clones of PMAD16::pSDH156, 12 clones of PMAD18::pSDH156, 6 clones of PMAD16::pSDH160 and 6 clones of PMAD18::pSDH160 were picked for follow-up western analysis. All clones were grown in the following manner: 5 ml cultures of each were inoculated using one colony of each strain in YEPD media (Per liter: 20.0 g D-Glucose (J. T. Baker), 20.0 g Bacto Peptone (Difco), 10.0 g Yeast Extract (Difco), 0.04 g adenine (Alfa Aesar), 0.06 g L-Leucine (TCI America) and water). Cultures were grown in test tubes and placed on a roller drum at 30° C. overnight. The following day, 0.5 ml of each overnight inoculum culture was diluted into 24.5 mls of BMY.1 media (Per liter: 13.4 g Yeast Nitrogen Base without amino acids (Becton Dickinson), 10.0 g Yeast Extract (Difco), 10.0 g tryptone (Difco), 10.0 g casamino acids (Difco), 6.7 g K2HPO4 (EM Science), 4.2 g citric acid (EM Science), and water) supplemented with 10 mls per liter of media with FXIII vitamin solution (0.05 g/L biotin, 0.8 g/L thiamine hydrochloride, 0.8 g/L pyroxidine HCL, 15.0 g/L inositol, 15.0 g/L calcium pantothenate, 0.6 g/L niacinamide, 0.1 g/L folic acid, 0.2g/L riboflavin, 1.0 g/L choline chloride) and 10 mls per liter of Methanol (J. T. Baker) for a 1% Methanol final concentration. Cultures were grown in 125 ml baffled flasks on a platform shaker set to 250 rpm at 30° C. for 48 hours. After 24 hours, a sample was taken for western analysis, and a 1% Methanol dose was added to each culture.
[0151]After 48 hours of growth and induction, the cultures were harvested at 10,000 rpm for 10 minutes in a Beckman centrifuge (JA-20 rotor) to pellet the cells. Two hundred fifty μl of zsig37 containing supernatant was mixed with 250 μl of 2× Laemmli Sample Buffer (125 mM Tris (Millipore), 20% glycerol (EM Science), 4% SDS (ICN), 0.01% Bromophenol blue (EM Science) and water). Each 500 μl sample was split into two 250 μl samples: one set of samples was treated with 2% β-mercaptoethanol (Sigma) and represents a reduced sample, while the other set represents the non-reduced sample.
[0152]An SDS-PAGE analysis was carried out as described below. All reduced samples were heated for 10 min at 65° C., while all non-reduced samples were not heated. Fifteen μL of each sample was applied for electrophoresis on a polyacrylamide gel. Protein separation was performed by electrophoresis in a 4-12% gradient Tris-Gly polyacrylamide resolving gel (Invitrogen) under denaturing conditions (SDS-PAGE) using lx Glycine running buffer (Invitrogen). The voltage of 80V was applied for the first 30 minutes, then the voltage was raised to 130V for the duration of the run. Subsequently, electrotransference was carried out to a 0.2 μm nitrocellulose membrane (Invitrogen) for 2 h at 200 mA (constant current). The blots were then developed as above.
[0153]The electrophoretic analysis on the polyacrylamide gel of the culture medium from P. methanolica clones representing pSDH156 (S. cerevisiae alpha factor D->Y pre-pro sequence) and pSDH160 (β-glucanase leader) showed that in the culture medium from both host strains a milieu appears corresponding to various zsig37 forms, while in the non-induced cell culture medium, there was no band. Roughly ninety percent of the recombinant clones that were analyzed for the integrated heterologous gene expression produced and secreted recombinant zsig37. The resulting zsig37 plasmid-containing yeast strains show the heterologous S. cerevisiae αFpp construct pSDH156 secretes equivalent levels of zsig37 compared to the endogenous P. methanolica quadrature-glucanase leader pSDH160 in the PMAD16 host strain background. Interestingly, plasmid-containing yeast strains show the endogenous P. methanolica β-glucanase leader construct pSDH160 secretes approximately 2-3 fold higher levels of zsig37 in PMAD16 compared to the PMAD18 host strain background. Every isolet of each αFpp::zsig37 strain was picked for subsequent use; the resulting clones were designated PMAD16::pSDH156 isolets #40, 56, 58, 84, 92, 149, 167, 169, 230, 231, and PMAD18::pSDH156 isolets #23, 29, 35, 144, 149, 161, 191, 202, 206, 217, 224, 269, respectively. In addition, every isolet of each β-glucanase::zsig37 strain was picked for subsequent use; the resulting clones were designated PMAD16::pSDH160 isolets #1, 2, 26, 30, 44, and PMAD18::pSDH160 isolets #1, 10, 21, 43, 48, 62, respectively.
[0154]The complete disclosure of all patents, patent applications, and publications, and electronically available material (e.g., GenBank amino acid and nucleotide sequence submissions) cited herein are incorporated by reference. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.
Sequence CWU
1
40184DNAPichia methanolicaCDS(1)...(84) 1atg aag ttc tcg cta agt aca ttg
aca gtt atc acc acc tta cta tca 48Met Lys Phe Ser Leu Ser Thr Leu
Thr Val Ile Thr Thr Leu Leu Ser 1 5 10
15ttg gtc tca gct gca cca ctc act ttg aaa aag aga
84Leu Val Ser Ala Ala Pro Leu Thr Leu Lys Lys Arg
20 25228PRTPichia methanolica 2Met Lys Phe Ser Leu Ser
Thr Leu Thr Val Ile Thr Thr Leu Leu Ser 1 5
10 15Leu Val Ser Ala Ala Pro Leu Thr Leu Lys Lys Arg
20 25384DNAArtificial SequenceDegenerate
polynucleotide encoding SEQ ID NO2 3atgaarttyw snytnwsnac nytnacngtn
athacnacny tnytnwsnyt ngtnwsngcn 60gcnccnytna cnytnaaraa rmgn
8443077DNAPichia methanolica
4cagctgctct gctccttgat tcgtaattaa tgttatcctt ttactttgaa ctcttgtcgg
60tccccaacag ggattccaat cggtgctcag cgggatttcc catgaggttt ttgacaactt
120tattgatgct gcaaaaactt ttttagccgg gtttaagtaa ctgggcaata tttccaaagg
180ctgtgggcgt tccacactcc ttgcttttca taatctctgt gtattgtttt attcgcattt
240tgattctctt attaccagtt atgtagaaag atcggcaaac aaaatatcaa cttttatctt
300gaacgctgac ccacggtttc aaataactat cagaactcta tagctatagg ggaagtttac
360tgcttgctta aagcggctaa aaagtgtttg gcaaattaaa aaagctgtga caagtaggaa
420ctcctgtaaa gggccgattc gacttcgaaa gagcctaaaa acagtgacta ttggtgacgg
480aaaattgcta aaggagtact agggctgtag taataaataa tggaacagtg gtacaacaat
540aaaagaatga cgctgtatgt cgtagcctgc acgagtagct cagtggtaga gcagcagatt
600gcaaatctgt tggtcaccgg ttcgatccgg tctcgggctt ccttttttgc tttttcgata
660tttgcgggta ggaagcaagg tctagttttc gtcgtttcgg atggtttacg aaagtatcag
720ccatgagtgt ttccctctgg ctacctaata tatttattga tcggtctctc atgtgaatgt
780ttctttccaa gttcggcttt cagctcgtaa atgtgcaaga aatatttgac tccagcgacc
840tttcagagtc aaattaattt tcgctaacaa tttgtgtttt tctggagaaa cctaaagatt
900taactgataa gtcgaatcaa catctttaaa tcctttagtt aagatctctg cagcggccag
960tattaaccaa tagcatattc acaggcatca catcggaaca ttcagaatgg actcgcaaac
1020tgtcgggatt ttaggtggtg gccaacttgg tcgtatgatc gttgaagctg cacacagatt
1080gaatatcaaa actgtgattc tcgaaaatgg agaccaggct ccagcaaagc aaatcaacgc
1140tttagatgac catattgacg gctcattcaa tgatccaaaa gcaattgccg aattggctgc
1200caagtgtgat gttttaaccg ttgagattga acatgttgac actgatgcgt tggttgaagt
1260tcaaaaggca actggcatca aaatcttccc atcaccagaa actatttcat tgatcaaaga
1320taaatacttg caaaaagagc atttgattaa gaatggcatt gctgttgccg aatcttgtag
1380tgttgaaagt agcgcagcat ctttagaaga agttggtgcc aaatacggct tcccatacat
1440gctaaaatct agaacaatgg cctatgacgg aagaggtaat tttgttgtca aagacaagtc
1500atatatacct gaagctttga aagttttaga tgacaggccg ttatacgccg agaaatgggc
1560tccattttca aaggagttag ctgttatggt tgtgagatca atcgatggcc aagtttattc
1620ctacccaact gttgaaacca tccaccaaaa caacatctgt cacactgtct ttgctccagc
1680tagagttaac gatactgtcc aaaagaaggc ccaaattttg gctgacaacg ctgtcaaatc
1740tttcccaggt gctggtatct ttggtgttga aatgttttta ttacaaaatg gtgacttatt
1800agtcaacgaa attgccccaa gacctcacaa ttctggtcac tataccatcg acgcttgtgt
1860cacctcgcaa tttgaagctc atgttagggc cattactggt ctacccatgc cgaagaactt
1920cacttgtttg tcgactccat ctacccaagc tattatgttg aacgttttag gtggcgatga
1980gcaaaacggt gagttcaaga tgtgtaaaag agcactagaa actcctcatg cttctgttta
2040cttatacggt aagactacaa gaccaggcag aaaaatgggt cacattaata tagtttctca
2100atcaatgact gactgtgagc gtagattaca ttacatagaa ggtacgacta acagcatccc
2160tctcgaagaa cagtacacta cagattccat tccgggcact tcaagcaagc cattagtcgg
2220tgtcatcatg ggttccgatt cggacctacc agtcatgtct ctaggttgta atatattgaa
2280gcaatttaac gttccatttg aagtcactat cgtttccgct catagaaccc cacaaagaat
2340ggccaagtat gccattgatg ctccaaagag agggttgaag tgcatcattg ctggtgctgg
2400tggtgccgct catttaccgg gaatggttgc ggcgatgacg ccgctgcctg ttattggtgt
2460ccctgttaaa ggctctactt tggatggtgt tgattcacta cactccatcg ttcaaatgcc
2520aagaggtatt cctgttgcta ctgtggctat taacaatgct actaacgctg ccttgctagc
2580tatcacaatc ttaggtgccg gcgatccaaa tacttgtctg caatggaagt ttatatgaac
2640aatatggaaa atgaagtttt gggcaaggct gaaaaattgg aaaatggtgg atatgaagaa
2700tacttgagta catacaagaa gtagaacctt ttatatttga tatagtactt actcaaagtc
2760ttaattgttc taactgttaa tttctgcttt gcatttctga aaagtttaag acaagaaatc
2820ttgaaatttc tagttgctcg taagaggaaa cttgcattca aataacatta acaataaatg
2880acaataatat attatttcaa cactgctata tggtagtttt ataggtttgg ttaggatttg
2940agatattgct agcgcttatc attatcctta attgttcatc gacgcaaatc gacgcatttc
3000cacaaaaatt ttccgaacct gtttttcact tctccagatc ttggtttagt atagcttttg
3060acacctaata cctgcag
307754409DNAPichia methanolicaCDS(1733)...(2734) 5cccgggggat cttattttct
gcaagaactt aaccgaggga catgtcaaac caagcatact 60gtaaaagaaa tagccgatgg
tttatatata tatatacttg cgttagtaga aacagtttat 120gcatgcatgg atgcaagaac
tcagatatca ggttatcaag aaacatggag aaattcctaa 180acagaaacgg aattaatccg
aaattctcgg tctcccaaag aaaatagatg cacaagctaa 240tacagcttgc taactagctt
caactttcaa aaaaaattct aagctattga atattcatca 300agataatagt ctatataaag
atgtaaagtc attattattg ggatatataa acgtcctata 360tattgctgaa atgttaggtg
tatgtactga aaacaatcag tttgagttta ccagagagag 420acgatggatc tacagatcaa
tagagagaga ataagatgag aataagatga ttaatagtga 480gaggtagtag ccactggcgg
gaggatgaaa atatcccgga taaacttaga aagaaattaa 540ttacacgtat aggtaacatt
tgttattgtc gaatctcaga tcagttgatg cctggaacag 600atcgacttat agatattatc
agatcataat catgaggcga ggtgcgacta gtaccaggtg 660atgatatatt gtttccggtt
atttcaaata gttgacgtcg ttgtgtgatt gggaaggcgt 720cggagtaaca gaaacagtaa
cggtacaagc atcattatga gttgagggta tgtagggaag 780cagttgtttg taagcatgtt
tacaaatgca atgcatgtta cgattggact acaattaaat 840ccgaatgtac ctatataacg
tgttgtacgt gttgtgccgt aagtagcccg atactagatg 900cttactacgt cactgatctg
ttcggatctc agtccattca tgtgtcaaaa tagttagtag 960ctaaggggga tacagggaag
atgtttggta cgattatcgg agggatgtgt cttctgaggg 1020gggaggagag agggcgtgta
aggagtttgt ttgtttgttt gtttgttgag agaagggggg 1080gagaagaggg ggtggtgggc
tgatggcaat tgatatagag ggagagtgtg cgttaactgt 1140ttagtgtggt ggcggtacgg
ggtacactgt agagggggac attataatgg ttatgtgtat 1200atgctgtata tatgaataca
agtagggagt gactacacat tgcaattgat aatatgtgta 1260tgtgtgcgca tcagtatata
cactcggagg ttctgaaagc catcattgta ttggacgttt 1320gaatggtatt agatgacttg
ttgtactaga ggacggagaa tgggtgagtg gaagcaatag 1380ataataatgg aaagtttgct
cggtggtgga cattggcccg gagtagtgat accgtcacct 1440taaaattgca gttaggggat
gatgctccgg ggcacgacct gccaactaat ttaatagtcg 1500tctaacgctg gaacaggtgt
tgttccacaa gtagatgagt ttgttggttg gctggtcaaa 1560tgctgccttg atccatcgtt
ttatatataa agactcactt ctcctcctct tgttcaattg 1620tttcacactc aactgcttct
cccttatctt ttttttttcc ctgttttatt ccccattgaa 1680ctagatcaca tcttttcata
ttacacactt ttatttatta taattacaca aa atg gct 1738
Met Ala
1att aac gtt ggt att aac ggt ttc ggt aga atc
ggt aga tta gtc ttg 1786Ile Asn Val Gly Ile Asn Gly Phe Gly Arg Ile
Gly Arg Leu Val Leu 5 10 15aga
gtt gct tta tca aga aag gac atc aac att gtt gct gtc aat gat 1834Arg
Val Ala Leu Ser Arg Lys Asp Ile Asn Ile Val Ala Val Asn Asp 20
25 30cct ttc att gct gct gaa tac gct gct tac
atg ttc aag tac gat tcc 1882Pro Phe Ile Ala Ala Glu Tyr Ala Ala Tyr
Met Phe Lys Tyr Asp Ser 35 40 45
50act cac ggt aag tac gcc ggc gaa gtt tcc agt gac ggt aaa tac
tta 1930Thr His Gly Lys Tyr Ala Gly Glu Val Ser Ser Asp Gly Lys Tyr
Leu 55 60 65atc att gat
ggt aag aag att gaa gtt ttc caa gaa aga gac cca gtt 1978Ile Ile Asp
Gly Lys Lys Ile Glu Val Phe Gln Glu Arg Asp Pro Val 70
75 80aac atc cca tgg ggt aaa gaa ggt gtc caa
tac gtt att gac tcc act 2026Asn Ile Pro Trp Gly Lys Glu Gly Val Gln
Tyr Val Ile Asp Ser Thr 85 90
95ggt gtt ttc act acc ttg gct ggt gct caa aag cac att gat gcc ggt
2074Gly Val Phe Thr Thr Leu Ala Gly Ala Gln Lys His Ile Asp Ala Gly
100 105 110gct gaa aag gtt atc atc act
gct cca tct gct gat gct cca atg ttc 2122Ala Glu Lys Val Ile Ile Thr
Ala Pro Ser Ala Asp Ala Pro Met Phe115 120
125 130gtt gtt ggt gtt aac gaa aag gaa tac act tct gac
ttg aag att gtt 2170Val Val Gly Val Asn Glu Lys Glu Tyr Thr Ser Asp
Leu Lys Ile Val 135 140
145tct aac gct tca tgt acc acc aac tgt ttg gct cca tta gct aag gtt
2218Ser Asn Ala Ser Cys Thr Thr Asn Cys Leu Ala Pro Leu Ala Lys Val
150 155 160gtt aac gac aac ttt ggt
att gaa tca ggt tta atg acc act gtc cac 2266Val Asn Asp Asn Phe Gly
Ile Glu Ser Gly Leu Met Thr Thr Val His 165 170
175tcc att acc gct acc caa aag acc gtc gat ggt cca tca cac
aag gac 2314Ser Ile Thr Ala Thr Gln Lys Thr Val Asp Gly Pro Ser His
Lys Asp 180 185 190tgg aga ggt ggt aga
act gct tcc ggt aac att atc cca tca tct act 2362Trp Arg Gly Gly Arg
Thr Ala Ser Gly Asn Ile Ile Pro Ser Ser Thr195 200
205 210ggt gct gct aag gct gtt ggt aag gtt tta
cct gtc tta gct ggt aag 2410Gly Ala Ala Lys Ala Val Gly Lys Val Leu
Pro Val Leu Ala Gly Lys 215 220
225tta acc ggt atg tct tta aga gtt cct act acc gat gtt tcc gtt gtt
2458Leu Thr Gly Met Ser Leu Arg Val Pro Thr Thr Asp Val Ser Val Val
230 235 240gat tta acc gtt aac tta
aag act cca acc act tac gaa gct att tgt 2506Asp Leu Thr Val Asn Leu
Lys Thr Pro Thr Thr Tyr Glu Ala Ile Cys 245 250
255gct gct atg aag aag gct tct gaa ggt gaa tta aag ggt gtt
tta ggt 2554Ala Ala Met Lys Lys Ala Ser Glu Gly Glu Leu Lys Gly Val
Leu Gly 260 265 270tac act gaa gac gct
gtt gtt tcc act gat ttc tta acc gat aac aga 2602Tyr Thr Glu Asp Ala
Val Val Ser Thr Asp Phe Leu Thr Asp Asn Arg275 280
285 290tca tct atc ttt gat gct aag gct ggt atc
tta tta acc cca act ttc 2650Ser Ser Ile Phe Asp Ala Lys Ala Gly Ile
Leu Leu Thr Pro Thr Phe 295 300
305gtt aag tta atc tct tgg tac gat aac gaa tac ggt tac tcc acc aga
2698Val Lys Leu Ile Ser Trp Tyr Asp Asn Glu Tyr Gly Tyr Ser Thr Arg
310 315 320gtt gtt gat tta cta caa
cac gtt gct tcc gct taa atcttacaat 2744Val Val Asp Leu Leu Gln
His Val Ala Ser Ala * 325 330ctagattgtg
aagtataagt aagcaaaaat tatatatata tttgtctttc atagtataag 2804tatagttttc
atgagaaata cagataaaca acaaaaaata agttcttttt gaaaaagtta 2864gattttattc
ttgaacttag taaaagcctt ccttttacag ctgcttactt acaaccttga 2924aggctattgc
ataagctcaa ttgaaaacga gtataatata ctgatttcaa ggtttaatta 2984tctgtaattt
tcaagtactt ccatacgtgg aaacctccca caattaacag caacacgaaa 3044catccatcat
ccaacaaccg agatgcggat taggcccgga gagataatat ttttcggtgt 3104ggcggtggtt
tcaactccga acgcagcgca gccaaaagca aacagatgat ttagtgaact 3164cttcttatga
tagatttttg gctgattgag ttgatctgac ctgtgtggtt cgatcgaatt 3224ctattgtgtt
tgatgccctg gtagtggtgt gcttcatctt attgtgaagt gtgaatccta 3284gcgattatgg
catttggacg ccaactacta gctctgacgg tagtggcttc tacgaatgta 3344acttacaatt
ctgctcaatt cgaacatctt ttcagtaaga gaagttatat atgtatgtgt 3404gtatgtgtat
gtaaatatac ataaccgctt gtgggggtga tttttggttt gtactgatgt 3464gaaactcagt
gctatcggat gatgctgtca ccaacaacag ctgcttaacc ttctttttac 3524tattctgata
cagaattagg aaagtttccg gatttgtgat gtgcggcttt ggttgccatt 3584agtctccttt
ttttggaggg aggagtgaag tggtgcgtta tgtgccctga tccaatggtt 3644ttgaaagagg
gagctaggga tagttaatgg gtagacctat gaacattgtg tattaatata 3704ttgaaatata
caaacataac ggctgaaaac agcaagaaat caaaaaggca caatttcaat 3764ggtatataac
ttcaataatg atagtaatag taatggtagt agttattaca ggaggaataa 3824tatcaagaaa
ggaaaactaa aagtacacca acgtattcag aaatacaaaa acagcgaaca 3884aaatcgtcga
ttagtaattc atatcatgat tgccatccaa acagctttct ttcattgaac 3944tcacgagggc
ttgcactatt ttccctgctt gatgagtaat ccatcatttc aaactcggtt 4004gaacctgtag
caccagaagc gccatttgac gtaattggcc ttgtaatttg ctgttgttgt 4064tgggatatgt
ttgattcatt ttggaaacgt tcatgatgcc ctcttttttt gttgtttgtt 4124gttggtatcg
gtgaattcga tctagatgca gaactgccac tattgttgtt attgccgttg 4184ttcgcattat
tgttatcgtc aaagtcaaag tcaagtaatg gaagaccaag ggaagcatca 4244acaccaaaat
cattcaacat cagtaaatcc gagtacgact taatggtatc tgcctgaatc 4304gttgcttgct
gctgattatg ctgttgttgg ttttgttgtt gctgtttcgc agtcagttgg 4364aaatgatcca
ctagttctag agcggccgcc accgcggtgg agctc
440963333DNAPichia methanolicaCDS(1093)...(2094) 6cataaaccat aatagtataa
tttgttagac aagttcaaag aatttccaat aaaagtgtaa 60ttttcacatg catttcaacc
cggagaataa aattttaaga aatccgattg gatagtgtag 120aattattgtt catattgtgt
tataataatt gcaattaccc aacaaaactt gcattggtta 180gtcatcgtat ttcatgctat
tagctgaaag tagggtaatc gagcggtttg aatggctctg 240taaatctaaa ctctttatct
gaaatgtata ttagatccga catgatgcat ttggaggttc 300tgagaggtac cgcattgaat
ttctgtgtgg aattagatga gttgttgtac cagaagaggg 360aaaatgggca agtggtggca
atagtaaatt atgggaagta tggtggatat tggcccggcg 420tagtgacatc ctcaccttaa
aattgcctta ggggataatg tgccgggcac gtccagctaa 480ctaatttagt agtcgtctaa
aactggggaa catttgttgt tcctttgata gttatacgaa 540actgattgaa taaaaagttt
atattcttct tgatgatcct tctgtctaat tgatagaata 600ggaatttaga tagaaatatg
gaaatacaca aaatatatgt aataaaatca aaaggggaac 660aattcaaagg attcagcaat
caaaagggat gagtgattct gggtaataaa tgagcaataa 720attagtaata aattagtaac
aagttagtaa taaattagta ataaattagc aacaaatgaa 780caatagtaaa agctaaaaga
taaaacaaaa ggtaggagat aagcagtaaa gtccgaaagt 840aatcaggtga ctagagtaag
gatgagaatg aaggacagat tccttacagc tacataagta 900gatgagctgt tgacggtcag
atggtgcctt ggtccatggt ttcatatata aagaccctct 960tcgtctcctt ttgttcgctt
gtttcacact caactgtttc tgattttacc ttttttcccc 1020tgcttgattc ccccattgaa
tcagatcaag tgttttcata gaacccactt ttatttattt 1080tagttgcaca aa atg gcc
att aac gtt ggt att aac ggt ttc ggg aga atc 1131 Met Ala
Ile Asn Val Gly Ile Asn Gly Phe Gly Arg Ile 1
5 10ggc aga tta gtc ttg aga gtt gcc tta tcg aga aaa
gac atc aac gtc 1179Gly Arg Leu Val Leu Arg Val Ala Leu Ser Arg Lys
Asp Ile Asn Val 15 20 25gtt gct gtc
aac gat cct ttc att gct cct gat tac gct gct tac atg 1227Val Ala Val
Asn Asp Pro Phe Ile Ala Pro Asp Tyr Ala Ala Tyr Met 30
35 40 45ttc aag tac gat tcc act cac ggt
aag tac act ggt gaa gtt tca agt 1275Phe Lys Tyr Asp Ser Thr His Gly
Lys Tyr Thr Gly Glu Val Ser Ser 50 55
60gat ggt aaa tac tta atc att gat ggt aag aag att gaa gtt
ttc caa 1323Asp Gly Lys Tyr Leu Ile Ile Asp Gly Lys Lys Ile Glu Val
Phe Gln 65 70 75gaa aga gat
cca gcc aac atc cca tgg ggg aaa gaa ggt gtt cag tac 1371Glu Arg Asp
Pro Ala Asn Ile Pro Trp Gly Lys Glu Gly Val Gln Tyr 80
85 90gtt att gaa tcc act ggc gtt ttc acc acc ttg
gct ggt gct caa aag 1419Val Ile Glu Ser Thr Gly Val Phe Thr Thr Leu
Ala Gly Ala Gln Lys 95 100 105cac att
gat gct ggt gcg gaa aag gtt atc atc act gct cca tct tct 1467His Ile
Asp Ala Gly Ala Glu Lys Val Ile Ile Thr Ala Pro Ser Ser110
115 120 125gat gct cca atg ttt gtt gtt
ggt gtt aac gaa aag gaa tac act cct 1515Asp Ala Pro Met Phe Val Val
Gly Val Asn Glu Lys Glu Tyr Thr Pro 130
135 140gac ttg aag att gtt tca aat gcc tca tgt acc acc
aac tgc gtg gct 1563Asp Leu Lys Ile Val Ser Asn Ala Ser Cys Thr Thr
Asn Cys Val Ala 145 150 155aca
tta gct aaa gtt gtt gac gat aac ttt gga att gaa tct ggg tta 1611Thr
Leu Ala Lys Val Val Asp Asp Asn Phe Gly Ile Glu Ser Gly Leu 160
165 170atg acc gct gtt cac gcc att act gct
tcc caa aag atc gtc gat ggt 1659Met Thr Ala Val His Ala Ile Thr Ala
Ser Gln Lys Ile Val Asp Gly 175 180
185ccc tcc cac aag gac tgg aga ggt ggt aga acc gct tcc ggc aac att
1707Pro Ser His Lys Asp Trp Arg Gly Gly Arg Thr Ala Ser Gly Asn Ile190
195 200 205atc cca tca tca
act ggt gct gct aag gct gtt ggt aag gtt ttg cca 1755Ile Pro Ser Ser
Thr Gly Ala Ala Lys Ala Val Gly Lys Val Leu Pro 210
215 220gct tta gct ggc aag cta acc ggt atg tct
ata agg gtt cct act act 1803Ala Leu Ala Gly Lys Leu Thr Gly Met Ser
Ile Arg Val Pro Thr Thr 225 230
235gat gtt tcc gtt gct gat tta acc gtt aac tta aag act gct acc acc
1851Asp Val Ser Val Ala Asp Leu Thr Val Asn Leu Lys Thr Ala Thr Thr
240 245 250tac cag gaa att tgc gct gct
ata aag aag gct tct gaa ggt gaa tta 1899Tyr Gln Glu Ile Cys Ala Ala
Ile Lys Lys Ala Ser Glu Gly Glu Leu 255 260
265aag ggt att tta ggt tac act gaa gat gcc gtt gtt tca acc gac ttc
1947Lys Gly Ile Leu Gly Tyr Thr Glu Asp Ala Val Val Ser Thr Asp Phe270
275 280 285tta acc gat agc
aga tcg tct atc ttc gat gcc aaa gct ggt atc tta 1995Leu Thr Asp Ser
Arg Ser Ser Ile Phe Asp Ala Lys Ala Gly Ile Leu 290
295 300tta acc cca acc ttc gtt aag cta atc tct
tgg tac gat aac gaa tac 2043Leu Thr Pro Thr Phe Val Lys Leu Ile Ser
Trp Tyr Asp Asn Glu Tyr 305 310
315ggt tat tcc acc aga gtt gtt gac tta cta caa cat gtt gct tcc gcc
2091Gly Tyr Ser Thr Arg Val Val Asp Leu Leu Gln His Val Ala Ser Ala
320 325 330taa atcttccaac ctaaattgcg
aaatataagc aagcaaaaat tatatgtata 2144*tttgtcttcc attgcataag
tctatctttc ctgagaaata acaaaaatat gttcttttcg 2204agacacttaa gttttatttt
tgcccttagt acaaggcatc catttgcagt tgctgcttac 2264agccctgaag gctattgcat
cagcccaatt ggaaacaagt atagcatact gatttgaggg 2324tttaattatc tgtaatattc
aagtacttat atgcgtagaa cctccaaata gcaacacgaa 2384aatccatcat ccaacaatca
aagatgtgga gcaggccaag caagatgata ttttctcggt 2444ggtggcggtt tcaatttctg
gggtgcgtta ttgtgtggct tgtaccttgc agggtaaacc 2504ttcgccagca gttccagtgg
tctcttcgac gaacaacagg ctgaaattcg gctgtttcag 2564catggcttgt ttttcctcca
tgggactagc gtagatttat ccccccagaa agtttctctt 2624cttgaatatc tctggtaccg
accactaact agattataga ttactgcgac atgttaaagc 2684attgtcgggg tctttaagca
tgctcaacca acaggttgcc tgaagagctg cgtactaacc 2744tggaacaggg ttcacagaaa
gagggcaacc cagaaaaaac actatttgtt aacccttata 2804gtgaagagtg ggggtacaaa
atctttgacc cgtactccac tacgacagtt ttgataaaca 2864cttgcagatt acctaatttg
gtatgtacaa tttctaggca tgggataagt atagctttta 2924atccggaagg ttcggataaa
tactgtgctg tgtgccaggc aaatgcgtcc cactggagaa 2984aaaggtaaag ccgactaacc
gaagacccac ctacaataaa tttaccgagc caccgaaaaa 3044ctcacgttac tcaatatatg
agtaatgtac tactataact atgtgtggaa tagaattgta 3104ttgtatagta gctcagcttt
cttcctggta tacggtcgac tttagcctaa acacttgttg 3164gttcagtgaa tacagcctga
ttagactaaa aggtagaagg actataaagg tgtacatacg 3224gaaatcctac tccccactta
aatagacaaa acccctctaa gtgttgtttc gacgtaaagc 3284tttgtttact gacaagcctt
ggcaccgatc ccccgggctg caggaattc 3333720DNAArtificial
SequenceOligonucleotide ZC18176 7aaytcncgnt gggaytaygg
20820DNAArtificial SequenceOligonucleotide
ZC18177 8aaytcnagrt gggaytaygg
20921DNAArtificial SequenceOligonucleotide ZC16562 9ccctggggca
ccgtgcaagt c
211024DNAArtificial SequenceOligonucleotide ZC16567 10tcctgagtta
tcaaagccgt tttg
241120DNAArtificial SequenceOligonucleotide ZC18180 11tgnganccng
gnacnccrtg
201220DNAArtificial SequenceOligonucleotide ZC18181 12ccngarttrt
craanccrtt
20131696DNAHomo sapiensCDS(69)...(806) 13cccgaggaga ccacgctcct ggagctctgc
tgtcttctca gggagactct gaggctctgt 60tgagaatc atg ctt tgg agg cag ctc
atc tat tgg caa ctg ctg gct ttg 110 Met Leu Trp Arg Gln Leu
Ile Tyr Trp Gln Leu Leu Ala Leu 1 5
10ttt ttc ctc cct ttt tgc ctg tgt caa gat gaa tac atg gag tct cca
158Phe Phe Leu Pro Phe Cys Leu Cys Gln Asp Glu Tyr Met Glu Ser Pro 15
20 25 30caa acc gga gga
cta ccc cca gac tgc agt aag tgt tgt cat gga gac 206Gln Thr Gly Gly
Leu Pro Pro Asp Cys Ser Lys Cys Cys His Gly Asp 35
40 45tac agc ttt cga ggc tac caa ggc ccc cct
ggg cca ccg ggc cct cct 254Tyr Ser Phe Arg Gly Tyr Gln Gly Pro Pro
Gly Pro Pro Gly Pro Pro 50 55
60ggc att cca gga aac cat gga aac aat ggc aac aat gga gcc act ggt
302Gly Ile Pro Gly Asn His Gly Asn Asn Gly Asn Asn Gly Ala Thr Gly
65 70 75cat gaa gga gcc aaa ggt gag
aag ggc gac aaa ggt gac ctg ggg cct 350His Glu Gly Ala Lys Gly Glu
Lys Gly Asp Lys Gly Asp Leu Gly Pro 80 85
90cga ggg gag cgg ggg cag cat ggc ccc aaa gga gag aag ggc tac ccg
398Arg Gly Glu Arg Gly Gln His Gly Pro Lys Gly Glu Lys Gly Tyr Pro 95
100 105 110ggg att cca cca
gaa ctt cag att gca ttc atg gct tct ctg gca acc 446Gly Ile Pro Pro
Glu Leu Gln Ile Ala Phe Met Ala Ser Leu Ala Thr 115
120 125cac ttc agc aat cag aac agt ggg att atc
ttc agc agt gtt gag acc 494His Phe Ser Asn Gln Asn Ser Gly Ile Ile
Phe Ser Ser Val Glu Thr 130 135
140aac att gga aac ttc ttt gat gtc atg act ggt aga ttt ggg gcc cca
542Asn Ile Gly Asn Phe Phe Asp Val Met Thr Gly Arg Phe Gly Ala Pro
145 150 155gta tca ggt gtg tat ttc ttc
acc ttc agc atg atg aag cat gag gat 590Val Ser Gly Val Tyr Phe Phe
Thr Phe Ser Met Met Lys His Glu Asp 160 165
170gtt gag gaa gtg tat gtg tac ctt atg cac aat ggc aac aca gtc ttc
638Val Glu Glu Val Tyr Val Tyr Leu Met His Asn Gly Asn Thr Val Phe175
180 185 190agc atg tac agc
tat gaa atg aag ggc aaa tca gat aca tcc agc aat 686Ser Met Tyr Ser
Tyr Glu Met Lys Gly Lys Ser Asp Thr Ser Ser Asn 195
200 205cat gct gtg ctg aag cta gcc aaa ggg gat
gag gtt tgg ctg cga atg 734His Ala Val Leu Lys Leu Ala Lys Gly Asp
Glu Val Trp Leu Arg Met 210 215
220ggc aat ggc gct ctc cat ggg gac cac caa cgc ttc tcc acc ttt gca
782Gly Asn Gly Ala Leu His Gly Asp His Gln Arg Phe Ser Thr Phe Ala
225 230 235gga ttc ctg ctc ttt gaa act
aag taaatatatg actagaatag ctccactttg 836Gly Phe Leu Leu Phe Glu Thr
Lys 240 245gggaagactt gtagctgagc tgatttgtta cgatctgagg
aacattaaag ttgagggttt 896tacattgctg tattcaaaaa attattggtt gcaatgttgt
tcacgctaca ggtacaccaa 956taatgttgga caattcaggg gctcagaaga atcaaccaca
aaatagtctt ctcagatgac 1016cttgactaat atactcagca tctttatcac tctttccttg
gcacctaaaa gataattctc 1076ctctgacgca ggttggaaat atttttttct atcacagaag
tcatttgcaa agaattttga 1136ctactctgct tttaatttaa taccagtttt caggaacccc
tgaagtttta agttcattat 1196tctttataac atttgagaga atcggatgta gtgatatgac
agggctgggg caagaacagg 1256ggcactagct gccttattag ctaatttagt gccctccgtg
ttcagcttag cctttgaccc 1316tttccttttg atccacaaaa tacattaaaa ctctgaattc
acatacaatg ctattttaaa 1376gtcaatagat tttagctata aagtgcttga ccagtaatgt
ggttgtaatt ttgtgtatgt 1436tcccccacat cgcccccaac ttcggatgtg gggtcaggag
gttgaggttc actattaaca 1496aatgtcataa atatctcata gaggtacagt gccaatagat
attcaaatgt tgcatgttga 1556ccagagggat tttatatctg aagaacatac actattaata
aataccttag agaaagattt 1616tgacctggct ttagataaaa ctgtggcaag aaaaatgtaa
tgagcaatat atggaaataa 1676acacaccttt gttaaagata
169614246PRTHomo sapiens 14Met Leu Trp Arg Gln Leu
Ile Tyr Trp Gln Leu Leu Ala Leu Phe Phe 1 5
10 15Leu Pro Phe Cys Leu Cys Gln Asp Glu Tyr Met Glu
Ser Pro Gln Thr 20 25 30Gly
Gly Leu Pro Pro Asp Cys Ser Lys Cys Cys His Gly Asp Tyr Ser 35
40 45Phe Arg Gly Tyr Gln Gly Pro Pro Gly
Pro Pro Gly Pro Pro Gly Ile 50 55
60Pro Gly Asn His Gly Asn Asn Gly Asn Asn Gly Ala Thr Gly His Glu65
70 75 80Gly Ala Lys Gly Glu
Lys Gly Asp Lys Gly Asp Leu Gly Pro Arg Gly 85
90 95Glu Arg Gly Gln His Gly Pro Lys Gly Glu Lys
Gly Tyr Pro Gly Ile 100 105
110Pro Pro Glu Leu Gln Ile Ala Phe Met Ala Ser Leu Ala Thr His Phe
115 120 125Ser Asn Gln Asn Ser Gly Ile
Ile Phe Ser Ser Val Glu Thr Asn Ile 130 135
140Gly Asn Phe Phe Asp Val Met Thr Gly Arg Phe Gly Ala Pro Val
Ser145 150 155 160Gly Val
Tyr Phe Phe Thr Phe Ser Met Met Lys His Glu Asp Val Glu
165 170 175Glu Val Tyr Val Tyr Leu Met
His Asn Gly Asn Thr Val Phe Ser Met 180 185
190Tyr Ser Tyr Glu Met Lys Gly Lys Ser Asp Thr Ser Ser Asn
His Ala 195 200 205Val Leu Lys Leu
Ala Lys Gly Asp Glu Val Trp Leu Arg Met Gly Asn 210
215 220Gly Ala Leu His Gly Asp His Gln Arg Phe Ser Thr
Phe Ala Gly Phe225 230 235
240Leu Leu Phe Glu Thr Lys 2451565DNAArtificial
SequenceOligonucleotide ZC37475 15attgctgcta aagaagaagg tgtaagcttg
tacaagagac aagatgaata catggagtct 60ccaca
651668DNAArtificial
SequenceOligonucleotide ZC37474 16caaaaattat aaaaatatcc aaacaggcag
ccgaattcta ttacttagtt tcaaagagca 60ggaatcct
681767DNAArtificial
SequenceOligonucleotide ZC39207 17aaaaaatctt actattaatt tctcaaaaga
attcaaaaga atgaagttct cgctaagtac 60attgaca
671863DNAArtificial
SequenceOligonucleotide ZC39209 18gggaccacct tccattggca tgtattcttc
ttctctcttt ttcaaagtga gtggtgcagc 60tga
631965DNAArtificial
SequenceOligonucleotide ZC39208 19tcattggtct cagctgcacc actcactttg
aaaaagagac aagatgaata catggagtct 60ccaca
6520754DNAArtificial
SequencealphaFpp::zacrp3 fragment 20attgctgcta aagaagaagg tgtatcctta
tacaagagac aagatgaata catggagtct 60ccacaaaccg gaggactacc cccagactgc
agtaagtgtt gtcatggaga ctacagcttt 120cgaggctacc aaggcccccc tgggccaccg
ggccctcctg gcattccagg aaaccatgga 180aacaatggca acaatggagc cactggtcat
gaaggagcca aaggtgagaa gggcgacaaa 240ggtgacctgg ggcctcgagg ggagcggggg
cagcatggcc ccaaaggaga gaagggctac 300ccggggattc caccagaact tcagattgca
ttcatggctt ctctggcaac ccacttcagc 360aatcagaaca gtgggattat cttcagcagt
gttgagacca acattggaaa cttctttgat 420gtcatgactg gtagatttgg ggccccagta
tcaggtgtgt atttcttcac cttcagcatg 480atgaagcatg aggatgttga ggaagtgtat
gtgtacctta tgcacaatgg caacacagtc 540ttcagcatgt acagctatga aatgaagggc
aaatcagata catccagcaa tcatgctgtg 600ctgaagctag ccaaagggga tgaggtttgg
ctgcgaatgg gcaatggcgc tctccatggg 660gaccaccaac gcttctccac ctttgcagga
ttcctgctct ttgaaactaa gtaatagaat 720tcggctgcct gtttggatat ttttataatt
tttg 75421157DNAPichia methanolica
21aaaaaatctt actattaatt tctcaaaaga attcaaaaga atgaagttct cgctaagtac
60attgacagtt atcaccacct tactatcatt ggtctcagct gcaccactca ctttgaaaaa
120gagagaagaa gaatacatgc caatggaagg tggtccc
15722754DNAHomo sapiens 22tcattggtct cagctgcacc actcactttg aaaaagagac
aagatgaata catggagtct 60ccacaaaccg gaggactacc cccagactgc agtaagtgtt
gtcatggaga ctacagcttt 120cgaggctacc aaggcccccc tgggccaccg ggccctcctg
gcattccagg aaaccatgga 180aacaatggca acaatggagc cactggtcat gaaggagcca
aaggtgagaa gggcgacaaa 240ggtgacctgg ggcctcgagg ggagcggggg cagcatggcc
ccaaaggaga gaagggctac 300ccggggattc caccagaact tcagattgca ttcatggctt
ctctggcaac ccacttcagc 360aatcagaaca gtgggattat cttcagcagt gttgagacca
acattggaaa cttctttgat 420gtcatgactg gtagatttgg ggccccagta tcaggtgtgt
atttcttcac cttcagcatg 480atgaagcatg aggatgttga ggaagtgtat gtgtacctta
tgcacaatgg caacacagtc 540ttcagcatgt acagctatga aatgaagggc aaatcagata
catccagcaa tcatgctgtg 600ctgaagctag ccaaagggga tgaggtttgg ctgcgaatgg
gcaatggcgc tctccatggg 660gaccaccaac gcttctccac ctttgcagga ttcctgctct
ttgaaactaa gtaatagaat 720tcggctgcct gtttggatat ttttataatt tttg
75423930DNAArtificial SequencepSDH147 23atgagatttc
cttctatttt tactgctgtt ttattcgctg cttcctccgc tttagctgct 60ccagtcaaca
ctaccactga agatgaaacg gctcaaattc cagctgaagc tgtcatcggt 120tactctgatt
tagaaggtga tttcgatgtt gctgttttgc cattttccaa ctccaccaat 180aacggtttat
tgtttatcaa tactactatt gctagcattg ctgctaaaga agaaggtgta 240tccttataca
agagacaaga tgaatacatg gagtctccac aaaccggagg actaccccca 300gactgcagta
agtgttgtca tggagactac agctttcgag gctaccaagg cccccctggg 360ccaccgggcc
ctcctggcat tccaggaaac catggaaaca atggcaacaa tggagccact 420ggtcatgaag
gagccaaagg tgagaagggc gacaaaggtg acctggggcc tcgaggggag 480cgggggcagc
atggccccaa aggagagaag ggctacccgg ggattccacc agaacttcag 540attgcattca
tggcttctct ggcaacccac ttcagcaatc agaacagtgg gattatcttc 600agcagtgttg
agaccaacat tggaaacttc tttgatgtca tgactggtag atttggggcc 660ccagtatcag
gtgtgtattt cttcaccttc agcatgatga agcatgagga tgttgaggaa 720gtgtatgtgt
accttatgca caatggcaac acagtcttca gcatgtacag ctatgaaatg 780aagggcaaat
cagatacatc cagcaatcat gctgtgctga agctagccaa aggggatgag 840gtttggctgc
gaatgggcaa tggcgctctc catggggacc accaacgctt ctccaccttt 900gcaggattcc
tgctctttga aactaagtaa
93024309PRTArtificial SequencepSDH147 24Met Arg Phe Pro Ser Ile Phe Thr
Ala Val Leu Phe Ala Ala Ser Ser 1 5 10
15Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr
Ala Gln 20 25 30Ile Pro Ala
Glu Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe 35
40 45Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr
Asn Asn Gly Leu Leu 50 55 60Phe Ile
Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val65
70 75 80Ser Leu Tyr Lys Arg Gln Asp
Glu Tyr Met Glu Ser Pro Gln Thr Gly 85 90
95Gly Leu Pro Pro Asp Cys Ser Lys Cys Cys His Gly Asp
Tyr Ser Phe 100 105 110Arg Gly
Tyr Gln Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Ile Pro 115
120 125Gly Asn His Gly Asn Asn Gly Asn Asn Gly
Ala Thr Gly His Glu Gly 130 135 140Ala
Lys Gly Glu Lys Gly Asp Lys Gly Asp Leu Gly Pro Arg Gly Glu145
150 155 160Arg Gly Gln His Gly Pro
Lys Gly Glu Lys Gly Tyr Pro Gly Ile Pro 165
170 175Pro Glu Leu Gln Ile Ala Phe Met Ala Ser Leu Ala
Thr His Phe Ser 180 185 190Asn
Gln Asn Ser Gly Ile Ile Phe Ser Ser Val Glu Thr Asn Ile Gly 195
200 205Asn Phe Phe Asp Val Met Thr Gly Arg
Phe Gly Ala Pro Val Ser Gly 210 215
220Val Tyr Phe Phe Thr Phe Ser Met Met Lys His Glu Asp Val Glu Glu225
230 235 240Val Tyr Val Tyr
Leu Met His Asn Gly Asn Thr Val Phe Ser Met Tyr 245
250 255Ser Tyr Glu Met Lys Gly Lys Ser Asp Thr
Ser Ser Asn His Ala Val 260 265
270Leu Lys Leu Ala Lys Gly Asp Glu Val Trp Leu Arg Met Gly Asn Gly
275 280 285Ala Leu His Gly Asp His Gln
Arg Phe Ser Thr Phe Ala Gly Phe Leu 290 295
300Leu Phe Glu Thr Lys30525759DNAArtificial SequencepSDH149
25atgaagttct cgctaagtac attgacagtt atcaccacct tactatcatt ggtctcagct
60gcaccactca ctttgaaaaa gagacaagat gaatacatgg agtctccaca aaccggagga
120ctacccccag actgcagtaa gtgttgtcat ggagactaca gctttcgagg ctaccaaggc
180ccccctgggc caccgggccc tcctggcatt ccaggaaacc atggaaacaa tggcaacaat
240ggagccactg gtcatgaagg agccaaaggt gagaagggcg acaaaggtga cctggggcct
300cgaggggagc gggggcagca tggccccaaa ggagagaagg gctacccggg gattccacca
360gaacttcaga ttgcattcat ggcttctctg gcaacccact tcagcaatca gaacagtggg
420attatcttca gcagtgttga gaccaacatt ggaaacttct ttgatgtcat gactggtaga
480tttggggccc cagtatcagg tgtgtatttc ttcaccttca gcatgatgaa gcatgaggat
540gttgaggaag tgtatgtgta ccttatgcac aatggcaaca cagtcttcag catgtacagc
600tatgaaatga agggcaaatc agatacatcc agcaatcatg ctgtgctgaa gctagccaaa
660ggggatgagg tttggctgcg aatgggcaat ggcgctctcc atggggacca ccaacgcttc
720tccacctttg caggattcct gctctttgaa actaagtaa
75926252PRTArtificial SequencepSDH149 26Met Lys Phe Ser Leu Ser Thr Leu
Thr Val Ile Thr Thr Leu Leu Ser 1 5 10
15Leu Val Ser Ala Ala Pro Leu Thr Leu Lys Lys Arg Gln Asp
Glu Tyr 20 25 30Met Glu Ser
Pro Gln Thr Gly Gly Leu Pro Pro Asp Cys Ser Lys Cys 35
40 45Cys His Gly Asp Tyr Ser Phe Arg Gly Tyr Gln
Gly Pro Pro Gly Pro 50 55 60Pro Gly
Pro Pro Gly Ile Pro Gly Asn His Gly Asn Asn Gly Asn Asn65
70 75 80Gly Ala Thr Gly His Glu Gly
Ala Lys Gly Glu Lys Gly Asp Lys Gly 85 90
95Asp Leu Gly Pro Arg Gly Glu Arg Gly Gln His Gly Pro
Lys Gly Glu 100 105 110Lys Gly
Tyr Pro Gly Ile Pro Pro Glu Leu Gln Ile Ala Phe Met Ala 115
120 125Ser Leu Ala Thr His Phe Ser Asn Gln Asn
Ser Gly Ile Ile Phe Ser 130 135 140Ser
Val Glu Thr Asn Ile Gly Asn Phe Phe Asp Val Met Thr Gly Arg145
150 155 160Phe Gly Ala Pro Val Ser
Gly Val Tyr Phe Phe Thr Phe Ser Met Met 165
170 175Lys His Glu Asp Val Glu Glu Val Tyr Val Tyr Leu
Met His Asn Gly 180 185 190Asn
Thr Val Phe Ser Met Tyr Ser Tyr Glu Met Lys Gly Lys Ser Asp 195
200 205Thr Ser Ser Asn His Ala Val Leu Lys
Leu Ala Lys Gly Asp Glu Val 210 215
220Trp Leu Arg Met Gly Asn Gly Ala Leu His Gly Asp His Gln Arg Phe225
230 235 240Ser Thr Phe Ala
Gly Phe Leu Leu Phe Glu Thr Lys 245
250271026DNAHomo sapiensCDS(1)...(1026) 27atg aga ttt cct tct att ttt act
gct gtt tta ttc gct gct tcc tcc 48Met Arg Phe Pro Ser Ile Phe Thr
Ala Val Leu Phe Ala Ala Ser Ser 1 5 10
15gct tta gct gct cca gtc aac act acc act gaa gat gaa acg
gct caa 96Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr
Ala Gln 20 25 30att cca gct
gaa gct gtc atc ggt tac tct gat tta gaa ggt gat ttc 144Ile Pro Ala
Glu Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe 35
40 45gat gtt gct gtt ttg cca ttt tcc aac tcc acc
aat aac ggt tta ttg 192Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr
Asn Asn Gly Leu Leu 50 55 60ttt atc
aat act act att gct agc att gct gct aaa gaa gaa ggt gta 240Phe Ile
Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val 65
70 75 80agc ttg tac aag aga aga gtt
cct cat gtc caa ggt gaa caa caa gag 288Ser Leu Tyr Lys Arg Arg Val
Pro His Val Gln Gly Glu Gln Gln Glu 85
90 95tgg gag ggt act gag gag ttg cca tcc cct cca gac cat
gcc gag aga 336Trp Glu Gly Thr Glu Glu Leu Pro Ser Pro Pro Asp His
Ala Glu Arg 100 105 110gct gaa
gaa caa cat gaa aaa tac aga cca tct caa gac caa ggt ttg 384Ala Glu
Glu Gln His Glu Lys Tyr Arg Pro Ser Gln Asp Gln Gly Leu 115
120 125cct gct tcc aga tgc ttg aga tgc tgt gac
cct ggt acc tcc atg tac 432Pro Ala Ser Arg Cys Leu Arg Cys Cys Asp
Pro Gly Thr Ser Met Tyr 130 135 140cca
gct acc gcc gtt cca caa atc aac atc act atc ttg aaa ggt gag 480Pro
Ala Thr Ala Val Pro Gln Ile Asn Ile Thr Ile Leu Lys Gly Glu145
150 155 160aag ggt gac aga gga gat
aga ggc ttg caa ggt aag tat ggc aaa aca 528Lys Gly Asp Arg Gly Asp
Arg Gly Leu Gln Gly Lys Tyr Gly Lys Thr 165
170 175ggc tca gca ggt gcc aga ggc cac act ggt cca aaa
ggt caa aag ggc 576Gly Ser Ala Gly Ala Arg Gly His Thr Gly Pro Lys
Gly Gln Lys Gly 180 185 190tcc
atg ggt gcc cct ggt gag aga tgc aag tcc cac tac gcc gcc ttt 624Ser
Met Gly Ala Pro Gly Glu Arg Cys Lys Ser His Tyr Ala Ala Phe 195
200 205tct gtt ggc aga aag aag cca atg cac
tcc aac cac tac tac caa act 672Ser Val Gly Arg Lys Lys Pro Met His
Ser Asn His Tyr Tyr Gln Thr 210 215
220gtt atc ttc gac act gag ttc gtt aac ttg tac gac cac ttc aac atg
720Val Ile Phe Asp Thr Glu Phe Val Asn Leu Tyr Asp His Phe Asn Met225
230 235 240ttc acc ggc aag
ttc tac tgc tac gtt cca ggc ttg tac ttc ttc tct 768Phe Thr Gly Lys
Phe Tyr Cys Tyr Val Pro Gly Leu Tyr Phe Phe Ser 245
250 255ttg aac gtt cac acc tgg aac caa aag gag
acc tac ctg cac atc atg 816Leu Asn Val His Thr Trp Asn Gln Lys Glu
Thr Tyr Leu His Ile Met 260 265
270aag aac gag gag gag gtt gtt atc ttg ttc gct caa gtt ggc gac aga
864Lys Asn Glu Glu Glu Val Val Ile Leu Phe Ala Gln Val Gly Asp Arg
275 280 285tct atc atg caa tct caa tct
ttg atg ctt gag ttg aga gag caa gac 912Ser Ile Met Gln Ser Gln Ser
Leu Met Leu Glu Leu Arg Glu Gln Asp 290 295
300caa gtt tgg gtt aga ttg tac aag ggc gaa cgt gag aac gcc atc ttc
960Gln Val Trp Val Arg Leu Tyr Lys Gly Glu Arg Glu Asn Ala Ile Phe305
310 315 320tct gag gag ttg
gac acc tac atc acc ttc tct ggc tac ttg gtc aag 1008Ser Glu Glu Leu
Asp Thr Tyr Ile Thr Phe Ser Gly Tyr Leu Val Lys 325
330 335cac gcc acc gag cca tag
1026His Ala Thr Glu Pro *
34028341PRTHomo sapiens 28Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe
Ala Ala Ser Ser 1 5 10
15Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln
20 25 30Ile Pro Ala Glu Ala Val Ile
Gly Tyr Ser Asp Leu Glu Gly Asp Phe 35 40
45Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu
Leu 50 55 60Phe Ile Asn Thr Thr Ile
Ala Ser Ile Ala Ala Lys Glu Glu Gly Val65 70
75 80Ser Leu Tyr Lys Arg Arg Val Pro His Val Gln
Gly Glu Gln Gln Glu 85 90
95Trp Glu Gly Thr Glu Glu Leu Pro Ser Pro Pro Asp His Ala Glu Arg
100 105 110Ala Glu Glu Gln His Glu
Lys Tyr Arg Pro Ser Gln Asp Gln Gly Leu 115 120
125Pro Ala Ser Arg Cys Leu Arg Cys Cys Asp Pro Gly Thr Ser
Met Tyr 130 135 140Pro Ala Thr Ala Val
Pro Gln Ile Asn Ile Thr Ile Leu Lys Gly Glu145 150
155 160Lys Gly Asp Arg Gly Asp Arg Gly Leu Gln
Gly Lys Tyr Gly Lys Thr 165 170
175Gly Ser Ala Gly Ala Arg Gly His Thr Gly Pro Lys Gly Gln Lys Gly
180 185 190Ser Met Gly Ala Pro
Gly Glu Arg Cys Lys Ser His Tyr Ala Ala Phe 195
200 205Ser Val Gly Arg Lys Lys Pro Met His Ser Asn His
Tyr Tyr Gln Thr 210 215 220Val Ile Phe
Asp Thr Glu Phe Val Asn Leu Tyr Asp His Phe Asn Met225
230 235 240Phe Thr Gly Lys Phe Tyr Cys
Tyr Val Pro Gly Leu Tyr Phe Phe Ser 245
250 255Leu Asn Val His Thr Trp Asn Gln Lys Glu Thr Tyr
Leu His Ile Met 260 265 270Lys
Asn Glu Glu Glu Val Val Ile Leu Phe Ala Gln Val Gly Asp Arg 275
280 285Ser Ile Met Gln Ser Gln Ser Leu Met
Leu Glu Leu Arg Glu Gln Asp 290 295
300Gln Val Trp Val Arg Leu Tyr Lys Gly Glu Arg Glu Asn Ala Ile Phe305
310 315 320Ser Glu Glu Leu
Asp Thr Tyr Ile Thr Phe Ser Gly Tyr Leu Val Lys 325
330 335His Ala Thr Glu Pro
3402964DNAArtificial SequenceZG42,210 primer 29attgctgcta aagaagaagg
tgtaagcttg tacaagagaa gagttcctca tgtccaaggt 60gaac
643061DNAArtificial
SequenceZG42,206 primer 30aaatatccaa acaggcagcc ctagaatact aggaattcta
tggctcggtg gcgtgcttga 60c
613167DNAArtificial SequenceZG42,209 primer
31aaaaaatctt actattaatt tctcaaaaga attcaaaaga atgaagttct cgctaagtac
60attgaca
673264DNAArtificial SequenceZG42,211 primer 32gttcaccttg gacatgagga
actcttctct ttttcaaagt gagtggtgca gctgagacca 60atga
643364DNAArtificial
SequenceZG42,273 primer 33tcattggtct cagctgcacc actcactttg aaaaagagaa
gagttcctca tgtccaaggt 60gaac
6434847DNAArtificial Sequencezsig37 sequence
amplified with ZG42,210 + ZG42,206 (pSDH156, or aFpp::zsig37)
34attgctgcta aagaagaagg tgtaagcttg tacaagagaa gagttcctca tgtccaaggt
60gaacaacaag agtgggaggg tactgaggag ttgccatccc ctccagacca tgccgagaga
120gctgaagaac aacatgaaaa atacagacca tctcaagacc aaggtttgcc tgcttccaga
180tgcttgagat gctgtgaccc tggtacctcc atgtacccag ctaccgccgt tccacaaatc
240aacatcacta tcttgaaagg tgagaagggt gacagaggag atagaggctt gcaaggtaag
300tatggcaaaa caggctcagc aggtgccaga ggccacactg gtccaaaagg tcaaaagggc
360tccatgggtg cccctggtga gagatgcaag tcccactacg ccgccttttc tgttggcaga
420aagaagccaa tgcactccaa ccactactac caaactgtta tcttcgacac tgagttcgtt
480aacttgtacg accacttcaa catgttcacc ggcaagttct actgctacgt tccaggcttg
540tacttcttct ctttgaacgt tcacacctgg aaccaaaagg agacctacct gcacatcatg
600aagaacgagg aggaggttgt tatcttgttc gctcaagttg gcgacagatc tatcatgcaa
660tctcaatctt tgatgcttga gttgagagag caagaccaag tttgggttag attgtacaag
720ggcgaacgtg agaacgccat cttctctgag gagttggaca cctacatcac cttctctggc
780tacttggtca agcacgccac cgagccatag aattcctagt attctagggc tgcctgtttg
840gatattt
847351026DNAArtificial SequencealphaFpp::zsig37 full-length nucleotide
sequence (pSDH156) 35atg aga ttt cct tct att ttt act gct gtt tta ttc
gct gct tcc tcc 48Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe
Ala Ala Ser Ser 1 5 10
15gct tta gct gct cca gtc aac act acc act gaa gat gaa acg gct caa
96Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln
20 25 30att cca gct gaa gct gtc atc
ggt tac tct gat tta gaa ggt gat ttc 144Ile Pro Ala Glu Ala Val Ile
Gly Tyr Ser Asp Leu Glu Gly Asp Phe 35 40
45gat gtt gct gtt ttg cca ttt tcc aac tcc acc aat aac ggt tta
ttg 192Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu
Leu 50 55 60ttt atc aat act act att
gct agc att gct gct aaa gaa gaa ggt gta 240Phe Ile Asn Thr Thr Ile
Ala Ser Ile Ala Ala Lys Glu Glu Gly Val 65 70
75 80agc ttg tac aag aga aga gtt cct cat gtc caa
ggt gaa caa caa gag 288Ser Leu Tyr Lys Arg Arg Val Pro His Val Gln
Gly Glu Gln Gln Glu 85 90
95tgg gag ggt act gag gag ttg cca tcc cct cca gac cat gcc gag aga
336Trp Glu Gly Thr Glu Glu Leu Pro Ser Pro Pro Asp His Ala Glu Arg
100 105 110gct gaa gaa caa cat gaa
aaa tac aga cca tct caa gac caa ggt ttg 384Ala Glu Glu Gln His Glu
Lys Tyr Arg Pro Ser Gln Asp Gln Gly Leu 115 120
125cct gct tcc aga tgc ttg aga tgc tgt gac cct ggt acc tcc
atg tac 432Pro Ala Ser Arg Cys Leu Arg Cys Cys Asp Pro Gly Thr Ser
Met Tyr 130 135 140cca gct acc gcc gtt
cca caa atc aac atc act atc ttg aaa ggt gag 480Pro Ala Thr Ala Val
Pro Gln Ile Asn Ile Thr Ile Leu Lys Gly Glu145 150
155 160aag ggt gac aga gga gat aga ggc ttg caa
ggt aag tat ggc aaa aca 528Lys Gly Asp Arg Gly Asp Arg Gly Leu Gln
Gly Lys Tyr Gly Lys Thr 165 170
175ggc tca gca ggt gcc aga ggc cac act ggt cca aaa ggt caa aag ggc
576Gly Ser Ala Gly Ala Arg Gly His Thr Gly Pro Lys Gly Gln Lys Gly
180 185 190tcc atg ggt gcc cct ggt
gag aga tgc aag tcc cac tac gcc gcc ttt 624Ser Met Gly Ala Pro Gly
Glu Arg Cys Lys Ser His Tyr Ala Ala Phe 195 200
205tct gtt ggc aga aag aag cca atg cac tcc aac cac tac tac
caa act 672Ser Val Gly Arg Lys Lys Pro Met His Ser Asn His Tyr Tyr
Gln Thr 210 215 220gtt atc ttc gac act
gag ttc gtt aac ttg tac gac cac ttc aac atg 720Val Ile Phe Asp Thr
Glu Phe Val Asn Leu Tyr Asp His Phe Asn Met225 230
235 240ttc acc ggc aag ttc tac tgc tac gtt cca
ggc ttg tac ttc ttc tct 768Phe Thr Gly Lys Phe Tyr Cys Tyr Val Pro
Gly Leu Tyr Phe Phe Ser 245 250
255ttg aac gtt cac acc tgg aac caa aag gag acc tac ctg cac atc atg
816Leu Asn Val His Thr Trp Asn Gln Lys Glu Thr Tyr Leu His Ile Met
260 265 270aag aac gag gag gag gtt
gtt atc ttg ttc gct caa gtt ggc gac aga 864Lys Asn Glu Glu Glu Val
Val Ile Leu Phe Ala Gln Val Gly Asp Arg 275 280
285tct atc atg caa tct caa tct ttg atg ctt gag ttg aga gag
caa gac 912Ser Ile Met Gln Ser Gln Ser Leu Met Leu Glu Leu Arg Glu
Gln Asp 290 295 300caa gtt tgg gtt aga
ttg tac aag ggc gaa cgt gag aac gcc atc ttc 960Gln Val Trp Val Arg
Leu Tyr Lys Gly Glu Arg Glu Asn Ala Ile Phe305 310
315 320tct gag gag ttg gac acc tac atc acc ttc
tct ggc tac ttg gtc aag 1008Ser Glu Glu Leu Asp Thr Tyr Ile Thr Phe
Ser Gly Tyr Leu Val Lys 325 330
335cac gcc acc gag cca tag
1026His Ala Thr Glu Pro * 34036341PRTArtificial
Sequenceb-glucanase::zsig37 full nucleotide sequence (pSDH160) 36Met
Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser 1
5 10 15Ala Leu Ala Ala Pro Val Asn
Thr Thr Thr Glu Asp Glu Thr Ala Gln 20 25
30Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly
Asp Phe 35 40 45Asp Val Ala Val
Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu 50 55
60Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu
Glu Gly Val65 70 75
80Ser Leu Tyr Lys Arg Arg Val Pro His Val Gln Gly Glu Gln Gln Glu
85 90 95Trp Glu Gly Thr Glu Glu
Leu Pro Ser Pro Pro Asp His Ala Glu Arg 100
105 110Ala Glu Glu Gln His Glu Lys Tyr Arg Pro Ser Gln
Asp Gln Gly Leu 115 120 125Pro Ala
Ser Arg Cys Leu Arg Cys Cys Asp Pro Gly Thr Ser Met Tyr 130
135 140Pro Ala Thr Ala Val Pro Gln Ile Asn Ile Thr
Ile Leu Lys Gly Glu145 150 155
160Lys Gly Asp Arg Gly Asp Arg Gly Leu Gln Gly Lys Tyr Gly Lys Thr
165 170 175Gly Ser Ala Gly
Ala Arg Gly His Thr Gly Pro Lys Gly Gln Lys Gly 180
185 190Ser Met Gly Ala Pro Gly Glu Arg Cys Lys Ser
His Tyr Ala Ala Phe 195 200 205Ser
Val Gly Arg Lys Lys Pro Met His Ser Asn His Tyr Tyr Gln Thr 210
215 220Val Ile Phe Asp Thr Glu Phe Val Asn Leu
Tyr Asp His Phe Asn Met225 230 235
240Phe Thr Gly Lys Phe Tyr Cys Tyr Val Pro Gly Leu Tyr Phe Phe
Ser 245 250 255Leu Asn Val
His Thr Trp Asn Gln Lys Glu Thr Tyr Leu His Ile Met 260
265 270Lys Asn Glu Glu Glu Val Val Ile Leu Phe
Ala Gln Val Gly Asp Arg 275 280
285Ser Ile Met Gln Ser Gln Ser Leu Met Leu Glu Leu Arg Glu Gln Asp 290
295 300Gln Val Trp Val Arg Leu Tyr Lys
Gly Glu Arg Glu Asn Ala Ile Phe305 310
315 320Ser Glu Glu Leu Asp Thr Tyr Ile Thr Phe Ser Gly
Tyr Leu Val Lys 325 330
335His Ala Thr Glu Pro 34037149DNAArtificial
Sequenceb-glucanase sequence amplified with ZG42,209 + ZG42,211
(b-blucanase::zsig37) 37aaaaaatctt actattaatt tctcaaaaga attcaaaaga
atgaagttct cgctaagtac 60attgacagtt atcaccacct tactatcatt ggtctcagct
gcaccactca ctttgaaaaa 120gagaagagtt cctcatgtcc aaggtgaac
14938847DNAArtificial Sequencezsig37 sequence
amplified with ZG42,273 + ZG42,206 (b-glucanase::zsig37)
38tcattggtct cagctgcacc actcactttg aaaaagagaa gagttcctca tgtccaaggt
60gaacaacaag agtgggaggg tactgaggag ttgccatccc ctccagacca tgccgagaga
120gctgaagaac aacatgaaaa atacagacca tctcaagacc aaggtttgcc tgcttccaga
180tgcttgagat gctgtgaccc tggtacctcc atgtacccag ctaccgccgt tccacaaatc
240aacatcacta tcttgaaagg tgagaagggt gacagaggag atagaggctt gcaaggtaag
300tatggcaaaa caggctcagc aggtgccaga ggccacactg gtccaaaagg tcaaaagggc
360tccatgggtg cccctggtga gagatgcaag tcccactacg ccgccttttc tgttggcaga
420aagaagccaa tgcactccaa ccactactac caaactgtta tcttcgacac tgagttcgtt
480aacttgtacg accacttcaa catgttcacc ggcaagttct actgctacgt tccaggcttg
540tacttcttct ctttgaacgt tcacacctgg aaccaaaagg agacctacct gcacatcatg
600aagaacgagg aggaggttgt tatcttgttc gctcaagttg gcgacagatc tatcatgcaa
660tctcaatctt tgatgcttga gttgagagag caagaccaag tttgggttag attgtacaag
720ggcgaacgtg agaacgccat cttctctgag gagttggaca cctacatcac cttctctggc
780tacttggtca agcacgccac cgagccatag aattcctagt attctagggc tgcctgtttg
840gatattt
84739855DNAArtificial Sequenceb-glucanase::zsig37 full nucleotide
sequence (pSDH160) 39atg aag ttc tcg cta agt aca ttg aca gtt atc acc
acc tta cta tca 48Met Lys Phe Ser Leu Ser Thr Leu Thr Val Ile Thr
Thr Leu Leu Ser 1 5 10
15ttg gtc tca gct gca cca ctc act ttg aaa aag aga aga gtt cct cat
96Leu Val Ser Ala Ala Pro Leu Thr Leu Lys Lys Arg Arg Val Pro His
20 25 30gtc caa ggt gaa caa caa gag
tgg gag ggt act gag gag ttg cca tcc 144Val Gln Gly Glu Gln Gln Glu
Trp Glu Gly Thr Glu Glu Leu Pro Ser 35 40
45cct cca gac cat gcc gag aga gct gaa gaa caa cat gaa aaa tac
aga 192Pro Pro Asp His Ala Glu Arg Ala Glu Glu Gln His Glu Lys Tyr
Arg 50 55 60cca tct caa gac caa ggt
ttg cct gct tcc aga tgc ttg aga tgc tgt 240Pro Ser Gln Asp Gln Gly
Leu Pro Ala Ser Arg Cys Leu Arg Cys Cys 65 70
75 80gac cct ggt acc tcc atg tac cca gct acc gcc
gtt cca caa atc aac 288Asp Pro Gly Thr Ser Met Tyr Pro Ala Thr Ala
Val Pro Gln Ile Asn 85 90
95atc act atc ttg aaa ggt gag aag ggt gac aga gga gat aga ggc ttg
336Ile Thr Ile Leu Lys Gly Glu Lys Gly Asp Arg Gly Asp Arg Gly Leu
100 105 110caa ggt aag tat ggc aaa
aca ggc tca gca ggt gcc aga ggc cac act 384Gln Gly Lys Tyr Gly Lys
Thr Gly Ser Ala Gly Ala Arg Gly His Thr 115 120
125ggt cca aaa ggt caa aag ggc tcc atg ggt gcc cct ggt gag
aga tgc 432Gly Pro Lys Gly Gln Lys Gly Ser Met Gly Ala Pro Gly Glu
Arg Cys 130 135 140aag tcc cac tac gcc
gcc ttt tct gtt ggc aga aag aag cca atg cac 480Lys Ser His Tyr Ala
Ala Phe Ser Val Gly Arg Lys Lys Pro Met His145 150
155 160tcc aac cac tac tac caa act gtt atc ttc
gac act gag ttc gtt aac 528Ser Asn His Tyr Tyr Gln Thr Val Ile Phe
Asp Thr Glu Phe Val Asn 165 170
175ttg tac gac cac ttc aac atg ttc acc ggc aag ttc tac tgc tac gtt
576Leu Tyr Asp His Phe Asn Met Phe Thr Gly Lys Phe Tyr Cys Tyr Val
180 185 190cca ggc ttg tac ttc ttc
tct ttg aac gtt cac acc tgg aac caa aag 624Pro Gly Leu Tyr Phe Phe
Ser Leu Asn Val His Thr Trp Asn Gln Lys 195 200
205gag acc tac ctg cac atc atg aag aac gag gag gag gtt gtt
atc ttg 672Glu Thr Tyr Leu His Ile Met Lys Asn Glu Glu Glu Val Val
Ile Leu 210 215 220ttc gct caa gtt ggc
gac aga tct atc atg caa tct caa tct ttg atg 720Phe Ala Gln Val Gly
Asp Arg Ser Ile Met Gln Ser Gln Ser Leu Met225 230
235 240ctt gag ttg aga gag caa gac caa gtt tgg
gtt aga ttg tac aag ggc 768Leu Glu Leu Arg Glu Gln Asp Gln Val Trp
Val Arg Leu Tyr Lys Gly 245 250
255gaa cgt gag aac gcc atc ttc tct gag gag ttg gac acc tac atc acc
816Glu Arg Glu Asn Ala Ile Phe Ser Glu Glu Leu Asp Thr Tyr Ile Thr
260 265 270ttc tct ggc tac ttg gtc
aag cac gcc acc gag cca tag 855Phe Ser Gly Tyr Leu Val
Lys His Ala Thr Glu Pro * 275
28040284PRTArtificial Sequenceb-glucanase::zsig37 full nucleotide
sequence (pSDH160) 40Met Lys Phe Ser Leu Ser Thr Leu Thr Val Ile Thr
Thr Leu Leu Ser 1 5 10
15Leu Val Ser Ala Ala Pro Leu Thr Leu Lys Lys Arg Arg Val Pro His
20 25 30Val Gln Gly Glu Gln Gln Glu
Trp Glu Gly Thr Glu Glu Leu Pro Ser 35 40
45Pro Pro Asp His Ala Glu Arg Ala Glu Glu Gln His Glu Lys Tyr
Arg 50 55 60Pro Ser Gln Asp Gln Gly
Leu Pro Ala Ser Arg Cys Leu Arg Cys Cys65 70
75 80Asp Pro Gly Thr Ser Met Tyr Pro Ala Thr Ala
Val Pro Gln Ile Asn 85 90
95Ile Thr Ile Leu Lys Gly Glu Lys Gly Asp Arg Gly Asp Arg Gly Leu
100 105 110Gln Gly Lys Tyr Gly Lys
Thr Gly Ser Ala Gly Ala Arg Gly His Thr 115 120
125Gly Pro Lys Gly Gln Lys Gly Ser Met Gly Ala Pro Gly Glu
Arg Cys 130 135 140Lys Ser His Tyr Ala
Ala Phe Ser Val Gly Arg Lys Lys Pro Met His145 150
155 160Ser Asn His Tyr Tyr Gln Thr Val Ile Phe
Asp Thr Glu Phe Val Asn 165 170
175Leu Tyr Asp His Phe Asn Met Phe Thr Gly Lys Phe Tyr Cys Tyr Val
180 185 190Pro Gly Leu Tyr Phe
Phe Ser Leu Asn Val His Thr Trp Asn Gln Lys 195
200 205Glu Thr Tyr Leu His Ile Met Lys Asn Glu Glu Glu
Val Val Ile Leu 210 215 220Phe Ala Gln
Val Gly Asp Arg Ser Ile Met Gln Ser Gln Ser Leu Met225
230 235 240Leu Glu Leu Arg Glu Gln Asp
Gln Val Trp Val Arg Leu Tyr Lys Gly 245
250 255Glu Arg Glu Asn Ala Ile Phe Ser Glu Glu Leu Asp
Thr Tyr Ile Thr 260 265 270Phe
Ser Gly Tyr Leu Val Lys His Ala Thr Glu Pro 275
280
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