# Patent

## Title
Method for high-level secretory production of protein

## Abstract

This invention provides a means for enabling high-level secretory production of proteins, in particular proteins having complicated structures such as antibodies, in host cells such as yeast cells. The invention also provides transformed yeast cells having the activated HAC1 gene and the RRBP1 gene and a method for enabling high-level secretory production of foreign proteins using such transformed host cells by inhibiting O-sugar chain formation indigenous to host cells such as yeast cells.

## Background
TECHNICAL FIELD
The present invention relates to a method for high-level secretory production of a protein, mainly in yeast.
BACKGROUND ART
To date, production of proteins for pharmaceutical applications via gene recombination techniques has mainly involved the use of animal cells or E. coli cells as hosts. E. coli cells enable the production of target proteins at low cost; however, E. coli cells cannot undergo modification typified by sugar chain modification, and inactive proteins are produced in the inclusion bodies. This requires a process of solubilization, and thus, E. coli cells are not suitable for complicated procedures for protein production. In contrast, animal cells enable the production of target proteins as active proteins. Disadvantageously, however, use of animal cells would remarkably increase production costs in terms of equipment and material costs, due to time-consuming operations of breeding and culture of animal cells.
Among proteins, antibodies have been used as pharmaceutical products for a long time. Since they were derived from sources other than humans, antibodies against the administered antibodies were newly produced. Thus, such antibodies could not be administered more than once, and use thereof was restricted. In recent years, production of a humanized antibody in which an amino acid sequence other than the antigen-binding site has been substituted with a sequence derived from a human antibody became possible. Further, production of a human antibody-producing mouse into which a human antibody gene has been introduced became possible. Thus, application of antibodies as pharmaceutical products became extensive. At present, such antibodies are produced by cultured cells, such as CHO cells, into which genes encoding hybridomas or antibodies have been introduced. Such production, however, is problematic in terms of cost, productivity, safety, and the like.
In recent years, production of proteins for pharmaceutical applications has been attempted with the use of yeast, for the purpose of complementing the drawbacks described above. However, substantially no such attempts have been put to practical use. Regarding antibodies having complicated structures, in particular, there are examples of expression of Fab, a single stranded antibody (ScFv), or the like (Biosci. Biotechnol. Biochem., 64: 2138-2144, 2000). Productivity, however, is very low in terms of a full-length antibody (Proc. Natl. Acad. Sci. U.S.A., 85: 8678-8682, 1988). An example of antibody production with the use of yeast (Pichia pastoris) that produces a mammalian N-binding sugar chain was reported recently (Nature Biotechnology, 24: 210-215, 2006), although this report does not refer to the yield. Thus, high-level production of antibodies in yeast is difficult. Causes thereof are considered to be insufficient secretion ability of yeast, degradation caused by protease, or the like.
As a means for resolving such problems, a method involving the use of a protease deficient strain was proposed (Enzyme and Microbial Technology, 26: 671-677, 2000; Protein Expr. Purif., 20: 485-491, 2000, Biosci. Biotechnol. Biochem., 66: 628-631, 2002). The inventors have also developed a method of using a protease, which is a protease A (PEP4), protease B (PRB1), or yapsin (YPS1) gene deficient strain, to inhibit degradation of an antibody (WO 2003/091431). As a method for improving protein productivity via gene introduction, a method for improving ScFv productivity by allowing coexpression of parts of molecular chaperones that assist formation of 3-dimensional structures of proteins on the endoplasmic reticulum, such as BiP (KAR2)/PDI, was reported (Nat. Biotechnol. 16: 773-777, 1998), although this method merely produces a fragment of a single-stranded antibody.
Also, many inducible or constitutive promoters have been developed and used for producing foreign proteins. When genes encoding foreign proteins are allowed to express at high levels with the use of potent promoters in cells or when proteins that are less likely to fold are produced, however, aggregation occurs in the endoplasmic reticulum (ER) and resulting proteins may be sometimes accumulated in cells. Further, secretory proteins and membrane proteins are translated into proteins, incorporated into the endoplasmic reticulum immediately thereafter, subjected to a given modification, and then transferred to the Golgi apparatus. In such a case, unfolded proteins may be sometimes accumulated in the endoplasmic reticulum for some reason. This is referred to as “endoplasmic reticulum stress.” Examples of causes for such endoplasmic reticulum stress include disturbance of modification (e.g., addition of a sugar chain or disulfide bond) that occurs in the endoplasmic reticulum and deteriorated transportation from the endoplasmic reticulum. Mammalian cells have an “unfold protein response (UPR)” mechanism as a means for reacting against such endoplasmic reticulum stress. For example, proteins accumulated in the endoplasmic reticulum are protected by inhibition of transcription, acceleration of folding induced by molecular chaperones, degradation of denatured proteins, or cell death via apoptosis.
As genes that regulate UPR, genes of IRE1α-XBP1, PERK-eIF-2α, and ATF6 animal cells are known. In case of yeast, Ire1p-Hac1p is the only gene that is known as such gene, and the Ire1p-Hac1p gene is associated with UPR by the mechanism shown below (see FIG. 1). First of all, Ire1p is generally bound to an antibody heavy chain binding protein (BiP). When an unfolded protein (UFP) is produced, however, BiP binds to such UFP. Ire1p dissociated from BiP is activated via autophosphorylation or dimerization, and it exhibits endonuclease activity. While the HAC1 gene is generally in an inactivated state, Ire1p having endonuclease activity subjects mRNA transcribed from the HAC1 gene to splicing and produces active Hac1p (Cell, 87: 405-413, 1996; Cell, 90: 1031-1039, 1997; the EMBO Journal, 18: 3119-3132, 1999). Such active Hac1p migrates to the nucleus, acts as a transcription factor, and promotes expression of genes encoding various proteins associated with a series of reactions referred to as UPR, e.g., associated sugar chain addition, protein folding, protein degradation (ER-associated degradation: ERAD), protein sorting, lipid metabolism, or the like (Cell, 101: 249-258, 2000).
Regarding an attempt to improve productivity of foreign proteins utilizing activated Hac1p, there is an example in which the gene encoding activated Hac1p of a filamentous bacterium, i.e., Trichoderma reesei, is introduced into S. cerevisiae to improve secretion of a heterogeneous protein, α-amylase, and an endogenous protein, i.e., invertase, (Appl. Environ. Microbiol. 69: 2065-2072, 2003). However, a single protein, α-amylase or invertase, has been known as a protein that is easily secreted, and improvement in the amount of production is as low as approximately two times the amount of production prior to the improvement. In recent years, it has been reported that an antibody fragment, Fab, was produced using Pichia pastoris in a strain into which the activated HAC1 gene had been solely introduced (Biotechnology and Bioengineering, 94: 353-361). Productivity improvement via introduction of the activated HAC1 gene is as low as approximately 1.3 times.
Meanwhile, an example in which the mammalian-derived RRBP1 (ribosome-binding protein1, ribosome receptor, p180 protein) gene is solely introduced into a yeast strain, so as to quintuple the amount of proteins (bovine pancreatic trypsin inhibitor (BPTI)) secreted is known (The Journal of Cell Biology, 146: 273-284, 1999). At first, the RRBP1 gene was isolated from a dog as a gene encoding a protein binding to the ribosome (Nature, 346: 540-544, 1990). RRBP1 has a molecular weight of 180 kDa and a special structure such that a sequence comprising 10 amino acid residues on the N-terminal side is repeated 54 times and this region is bound to a ribosome. RRBP1 is known to be involved in enlargement of membrane structure and in stabilization of mRNA (the Journal of Cell Biology, 130: 29-39, 1995; the Journal of Cell Biology, 156: 993-1001, 2002). A successful example in the aforementioned BPTI has a molecular weight of 6,500, which represents a very small peptide. Such results cannot always be applied to other high-molecular-weight proteins or protein aggregates composed of different proteins such as light-chain or heavy-chain of antibodies.
The protein O-mannosyltransferase (PMT) gene is known to be associated with formation of O-sugar chains that are inherent to yeast or mold. The PMT gene product is localized on the ER membrane and has activity of adding mannose to a hydroxyl residue of serine (Ser) or threonine (Thr) of a secretory protein (hereafter, such activity is referred to as PMT activity). Some proteins to which sugar chains had been added by PMT serve as primary components of the yeast strain wall as mannoproteins. When PMT activity is extremely lowered, the cell wall is known to become fragile and to affect the growth of cells.
Regarding the PMT genes, the existence of seven genes, i.e., PMT1, 2, 3, 4, 5, 6, and 7, is known in Saccharomyces cerevisiae (S. cerevisiae) (Biochim. Biophys. Acta., 1426: 297-307, 1999). The PMT gene is classified into three types; i.e., the PMT1 family, the PMT2 family, and the PMT4 family. It is known that PMT1p and PMT2p exhibit activity upon formation of heterodimers, and PMT4p exhibits activity upon formation of homodimers. Because of amino acid sequence homology and the like, it is said that PMT5p complements PMT1p, and that PMT3p complements PMT2p. PMT6p is highly homologous to PMT2p and PMT3p, although the type of composite that exhibits activity is not known. Also, each PMT protein is known to have selectivity for a substrate protein.
As PMT genes of other types of budding yeast, five genes highly homologous to the PMT1, 2, 4, 5, and 6 genes of S. cerevisiae in the case of Candida albicans (Mol. Microbiol., 55: 546-560, 2005), a gene highly homologous at least to the PMT4 gene of S. cerevisiae in the case of Cryptococcus neoformans (Eukaryot. Cell, 6: 222-234, 2007), and three genes (oma1, 2, and 4) highly homologous to the PMT1, 2, and 4 genes in the case of fission yeast, i.e., Schizosaccharomyces pombe, (Mol. Microbiol., 57: 156-170, 2005) have been discovered. Further, the existence of five genes that are highly homologous to the PMT1, 2, 4, 5, and 6 genes of S. cerevisiae was observed in methanol-assimilating yeast, Ogataea minuta (O. minuta).
The PMT gene is also found in mold. The PmtA gene and two other genes are found in Aspergillus nidulans, and the Pmt1 gene, which is highly homologous to the PMT2 gene of S. cerevisiae, is found in Trichoderma reesei (Curr. Genet., 43: 11-16, 2003).
PMT activity is said to have effects of acting on a peptide hydrophobic region, enhancing peptide hydrophilicity, and inhibiting peptide aggregation in ER cavity. When foreign proteins are produced, however, PMT activity occasionally adds an unnecessary O-sugar chain, which may result in insufficient formation of protein composites, lowered activity, or the like. For multimeric proteins, such as antibodies, in particular, formation of aggregates thereof (which refers to formation of light chain and heavy chain aggregates, in the case of antibodies) may be inhibited.
JP Patent No. 3630424 and JP Patent Publication (kohyo) No. H08-509867 (A) (1996) propose a method for producing a recombinant protein via inhibition of O-sugar chain addition resulting from modification of the PMT gene. These patent documents, however, do not describe the formation of aggregates of a light-chain and a heavy-chain of antibody.
An example in which PMT1 and PMT2 gene-deficient strains associated with formation of O-sugar chains are used to inhibit addition of O-sugar chains to promote aggregation of antibody light-chain and heavy-chain molecules by approximately 1.5 times is provided in WO 2002/046437. This data is the result of a pulse-labeling experiment using an RI-labeled amino acid, but it is not the result of observing the entire culture process. Also, a degree of inhibition of sugar chain addition is further lowered, and antibody productivity is deteriorated.
Although the HAC1 gene induces UPR, some of the UPR-inducible genes are known to be PMT genes that add yeast-specific O-sugar chains (Cell, 101: 249-258, 2000). Accordingly, introduction of the HAC1 gene may not be sufficient to produce high-quality multimeric proteins, such as antibodies.
As described above, a variety of methods have been proposed as methods for high-level secretory production of proteins in yeast. However, substantially no method is sufficient at a practical level. A method for efficiently producing proteins, in particular, high-molecular-weight proteins or protein aggregates, including antibodies, has not yet been discovered. When a trait is introduced into a cell via gene introduction, gene destruction, or the like, in general, the cell would experience a given sort of stress. Thus, other modification may be provided, or an opposite action may occur. When high-level protein expression is intended, for example, UPR becomes activated, which results in a negative element, such as sugar chain modification, degradation by a proteasome, or ER-associated degradation (ERAD). Accordingly, high-level secretory production of proteins having complicated structures, such as antibodies, are not achieved by a single process, such as introduction of a single gene. Also, mere combination of several conventional methods would not always yield synergistic effects.
Accordingly, the present invention is intended to provide a method for high-level secretory production of proteins and, more particularly, proteins having complicated structures, such as antibodies, in yeast or other host cells.
DISCLOSURE OF THE INVENTION
The present inventors have conducted concentrated studies in order to attain the above object. As a result, they discovered that genes associated with high-level secretory production of proteins, i.e., the activated HAC1 (the Hac1 protein, which is a transcription factor induced by splicing of mRNA by Ire1p upon application of endoplasmic reticulum stress) gene and the RRBP1 (ribosome-binding protein 1, ribosome receptor, p180 protein) gene, are coexpressed in methanol-assimilating yeast, Ogataea minuta, and the amount of antibody secretory production can be increased by approximately 10 times. Further, they discovered that activity of protein O-mannosyltransferase (PMT) associated with O-sugar chain addition to a yeast-specific protein, which inhibits aggregation of heteromultimers such as antibodies, may be inhibited to further improve productivity.
Also, they discovered that the gene associated with high-level secretory production of a protein, i.e., the activated HAC1 gene, may be expressed, and activity of protein O-mannosyltransferase (PMT) associated with O-sugar chain addition to a yeast-specific protein, which inhibits aggregation of heteromultimers such as antibodies, may be inhibited to realize synergistic improvement in productivity.
The present invention has been completed upon such findings (see FIG. 2).
Specifically, the present invention includes the following inventions.
[1] A transformed host cell comprising the activated HAC1 gene and the RRBP1 gene.
[2] The transformed host cell according to [1], which comprises the activated HAC1 gene (1) and the RRBP1 gene (2) below:
(1) the activated HAC1 gene selected from among (a) to (d) below:
(a) a gene encoding a protein which consists of the amino acid sequence as shown in SEQ ID NO: 23;
(b) a gene encoding a protein which consists of an amino acid sequence having at least 70% homology with the amino acid sequence as shown in SEQ ID NO: 23 and has the function of activating the unfolded protein response (UPR);
(c) a gene encoding a protein which consists of an amino acid sequence derived from the amino acid sequence as shown in SEQ ID NO: 23 by deletion, substitution, and/or addition of one or several amino acids and has the function of activating UPR; and
(d) a gene which hybridizes under stringent conditions to a DNA consisting of the nucleotide sequence as shown in SEQ ID NO: 22 or a complementary nucleotide sequence thereof and encodes a protein having the function of activating UPR; and
(2) the RRBP1 gene selected from among (e) to (h) below:
(e) a gene encoding human- or dog-derived RRBP1;
(f) a gene encoding a protein which consists of an amino acid sequence having at least 70% homology with the amino acid sequence of human- or dog-derived RRBP1 and has ribosome-binding activity;
(g) a gene encoding a protein which consists of an amino acid sequence derived from the amino acid sequence human- or dog-derived RRBP1 by deletion, substitution, and/or addition of one or several amino acids and has ribosome-binding activity; and
(h) a gene which hybridizes under stringent conditions to a gene consisting of a nucleotide sequence of the human- or dog-derived RRBP1 gene or a complementary nucleotide sequence thereof and encodes a protein having ribosome-binding activity.
[3] The transformed cell according to [1] comprising the activated HAC1 gene (1) and the RRBP1 gene (2) below:
(1) the activated HAC1 gene selected from among (i) to (l) below:
(i) a gene encoding the activated HAC1 protein of Saccharomyces cerevisiae, Trichoderma reesei, or Aspergillus nidulans; 
(j) a gene encoding a protein which consists of an amino acid sequence having at least 70% homology with the amino acid sequence of the activated HAC1 protein of Saccharomyces cerevisiae, Trichoderma reesei, or Aspergillus nidulans and has the function of activating UPR;
(k) a gene encoding a protein which consists of an amino acid sequence derived from the amino acid sequence of the activated HAC1 protein of Saccharomyces cerevisiae, Trichoderma reesei, or Aspergillus nidulans by deletion, substitution, and/or addition of one or several amino acids and has the function of activating UPR; and
(l) a gene which hybridizes under stringent conditions to a gene consisting of a nucleotide sequence of the gene encoding the activated HAC1 protein of Saccharomyces cerevisiae, Trichoderma reesei, or Aspergillus nidulans or a complementary nucleotide sequence thereof and encodes a protein having the function of activating UPR; and
(2) the RRBP1 gene selected from among (e) to (h) below:
(e) a gene encoding human- or dog-derived RRBP1;
(f) a gene encoding a protein which consists of an amino acid sequence having at least 70% homology with the amino acid sequence of human- or dog-derived RRBP1 and has ribosome-binding activity;
(g) a gene encoding a protein which consists of an amino acid sequence derived from the amino acid sequence human- or dog-derived RRBP1 by deletion, substitution, and/or addition of one or several amino acids and has ribosome-binding activity; and
(h) a gene which hybridizes under stringent conditions to a gene consisting of a nucleotide sequence of the human- or dog-derived RRBP1 gene or a complementary nucleotide sequence thereof and encodes a protein having ribosome-binding activity.
[4] The transformed host cell according to any of [1] to [3], wherein the host cell is a eukaryotic cell.
[5] The transformed host cell according to [4], wherein the eukaryotic cell is yeast.
[6] The transformed host cell according to [5], wherein the yeast is methanol-assimilating yeast.
[7] The transformed host cell according to [6], wherein the methanol-assimilating yeast is Ogataea minuta. 
[8] The transformed host cell according to [5], wherein the yeast is Saccharomyces cerevisiae. 
[9] The transformed host cell according to any of [1] to [8], which comprises a gene encoding a foreign protein introduced therein.
[10] The transformed host cell according to [9], wherein the foreign protein is a multimeric protein.
[11] The transformed host cell according to [10], wherein the multimeric protein is a heteromultimer.
[12] The transformed host cell according to [11], wherein the heteromultimer is an antibody or a functional fragment thereof.
[13] A method for producing a protein comprising culturing the transformed host cell according to any of [9] to [12] in a medium and sampling a target protein from the culture product.
[14] The method according to [13], wherein culture is conducted under conditions in which protein O-mannosyltransferase (PMT) activity is inhibited.
[15] The method according to [14], wherein protein O-mannosyltransferase (PMT) activity is inhibited with the addition of an inhibitor of PMT activity to the medium.
[16] A gene encoding the activated HAC1 protein of methanol-assimilating yeast.
[17] A gene selected from among (a) to (d) below:
(a) a gene encoding a protein which consists of the amino acid sequence as shown in SEQ ID NO: 23;
(b) a gene encoding a protein which consists of an amino acid sequence having at least 70% homology with the amino acid sequence as shown in SEQ ID NO: 23 and has the function of activating the unfolded protein response (UPR);
(c) a gene encoding a protein which consists of an amino acid sequence derived from the amino acid sequence as shown in SEQ ID NO: 23 by deletion, substitution, and/or addition of one or several amino acids and has the function of activating UPR; and
(d) a gene which hybridizes under stringent conditions to a DNA consisting of the nucleotide sequence as shown in SEQ ID NO: 22 or a complementary nucleotide sequence thereof and encodes a protein having the function of activating UPR.
[18] An expression vector comprising the gene according to [17].
[19] The expression vector according to [18], which is pOMexPGHy/Hac1.
[20] An expression vector comprising the activated HAC1 gene and the RRBP1 gene.
[21] The expression vector according to [20], wherein the activated HAC1 gene is the gene according to [17].
[22] The expression vector according to [20], wherein the activated HAC1 gene is a gene encoding the activated HAC1 protein of Saccharomyces cerevisiae, Trichoderma reesei, or Aspergillus nidulans or a homologous gene thereof.
[23] The vector according to [20], which is YEp351GAP-II-aHAC1/p180.
[24] The expression vector according to [20], wherein the RRBP1 gene is the human- or dog-derived RRBP1 gene or a homologous gene thereof.
[25] A transformed host cell into which the expression vector according to any of [18] to [24] has been introduced.
[26] A transformed host cell into which an expression vector comprising the activated HAC1 gene and an expression vector comprising the RRBP1 gene have been introduced.
[27] The transformed host cell according to [26], wherein the expression vector comprising the activated HAC1 gene is the expression vector according to [18] or [19].
[28] The transformed host cell according to [26], wherein the activated HAC1 gene is a gene encoding the activated HAC1 protein of Saccharomyces cerevisiae, Trichoderma reesei, or Aspergillus nidulans or a homologous gene thereof.
[29] The transformed cell according to [26], wherein the RRBP1 gene is the human- or dog-derived RRBP1 gene or a homologous gene thereof.
[30] A method for producing a transformed host cell comprising the steps of:
(A) introducing the activated HAC1 gene into a host cell; and
(B) introducing the RRBP1 gene into a host cell.
[31] The method according to [30], wherein the activated HAC1 gene is any of the following genes:
(1) the gene according to [17];
(2) a gene encoding the activated HAC1 protein of Saccharomyces cerevisiae, Trichoderma reesei, or Aspergillus nidulans; 
(3) a gene encoding a protein which consists of an amino acid sequence having at least 70% homology with the amino acid sequence of the activated HAC1 protein of Saccharomyces cerevisiae, Trichoderma reesei, or Aspergillus nidulans and has the function of activating UPR;
(4) a gene encoding a protein which consists of an amino acid sequence derived from the amino acid sequence of the activated HAC1 protein of Saccharomyces cerevisiae, Trichoderma reesei, or Aspergillus nidulans by deletion, substitution, and/or addition of one or several amino acids and has the function of activating UPR; and
(5) a gene which hybridizes under stringent conditions to a gene consisting of a nucleotide sequence a gene encoding the activated HAC1 protein of Saccharomyces cerevisiae, Trichoderma reesei, or Aspergillus nidulans or a complementary nucleotide sequence thereof and encodes a protein having the function of activating UPR.
[32] The method according to [30], wherein the RRBP1 gene is any of the following genes:
(1) a gene encoding the human- or dog-derived RRBP1;
(2) a gene encoding a protein which consists of an amino acid sequence having at least 70% homology with the amino acid sequence of human- or dog-derived RRBP1 and has ribosome-binding activity;
(3) a gene encoding a protein which consists of an amino acid sequence derived from the amino acid sequence human- or dog-derived RRBP1 by deletion, substitution, and/or addition of one or several amino acids and has ribosome-binding activity; and
(4) a gene which hybridizes under stringent conditions to a gene consisting of a nucleotide sequence of the human- or dog-derived RRBP1 gene or a complementary nucleotide sequence thereof and encodes a protein having ribosome-binding activity.
[33] The method according to any of [30] to [32], wherein the host cell is a eukaryotic cell.
[34] The method according to [33], wherein the eukaryotic cell is yeast.
[35] The method according to [34], wherein the yeast is methanol-assimilating yeast.
[36] The method according to [35], wherein the methanol-assimilating yeast is Ogataea minuta. 
[37] The method according to [34], wherein the yeast is Saccharomyces cerevisiae. 
[38] A gene selected from among (a) to (d) below:
(a) a gene encoding a protein which consists of the amino acid sequence as shown in SEQ ID NO: 70;
(b) a gene encoding a protein which consists of an amino acid sequence having at least 70% homology with the amino acid sequence as shown in SEQ ID NO: 70 and has the function of activating the unfolded protein response (UPR);
(c) a gene encoding a protein which consists of an amino acid sequence derived from the amino acid sequence as shown in SEQ ID NO: 70 by deletion, substitution, and/or addition of one or several amino acids and has the function of activating UPR; and
(d) a gene which hybridizes under stringent conditions to a DNA consisting of the nucleotide sequence as shown in SEQ ID NO: 69a complementary nucleotide sequence thereof and encodes a protein having the function of activating UPR.
[39] An expression vector comprising the gene according to [38].
[40] The expression vector according to [39], which is pOMexPGHy/PpHac1.
[41] A method for producing a protein comprising culturing a transformed cell into which the activated HAC1 gene and/or the RRBP1 gene and a gene encoding a foreign protein have been introduced in a medium under conditions in which O-sugar chain synthesis is inhibited and sampling a target protein from the culture product.
[42] The method for producing a protein according to [41], wherein O-sugar chain synthesis is inhibited by insertionally inactivating the PMT gene.
[43] The method for producing a protein according to [41], wherein O-sugar chain synthesis is inhibited by adding the inhibitor of PMT activity to the medium.
[44] The method for producing a protein according to [41], wherein O-sugar chain synthesis is inhibited by insertionally inactivating the PMT gene and by adding the inhibitor of PMT activity to the medium.
[45] The method for producing a protein according to [43] or [44], wherein the inhibitor of PMT activity is 5-[[3,4-(1-phenylmethoxy)phenyl]methylene]-4-oxo-2-thioxo-3-thiazolidineacetic acid or {(5Z)-4-oxo-5-[3-(1-phenylethoxy)-4-(2-phenylethoxy)benzylidene]-2-thioxo-1,3-thiazolidin-3-yl}acetic acid.
[46] A transformed host cell with the insertionally inactivated PMT gene and with the activated HAC1 gene introduced therein.
[47] The transformed cell according to [46], wherein the host cell is Ogataea minuta. 



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows a method for obtaining the activated HAC1 gene of O. minuta and the structure thereof.
FIG. 2 schematically shows the technical scope of the present invention.
FIG. 3 shows the structures of an expression vector for the activated HAC1 gene of O. minuta (pOMexPGHy/Hac1), an expression vector for the human RRBP1 gene (pOMexGP1A/p180), and an expression vector for the human antibody gene (pOMexGAT-G/Ab).
FIG. 4 shows the results of Western analysis of antibodies secreted in the culture supernatant of an antibody-producing yeast strain into which the activated HAC1 gene of O. minuta and the human RRBP1 gene have been introduced.
FIG. 5 shows a chart showing the results of measuring the amount of secreted antibodies of an antibody-producing yeast strain into which the activated HAC1 gene of O. minuta and the human RRBP1 gene have been introduced.
FIG. 6 shows the results of Western analysis of antibodies secreted in the culture supernatant of an antibody-producing yeast strain into which the activated HAC1 gene of O. minuta and the human RRBP1 gene have been introduced, which have been cultured under conditions in which O-sugar chain formation is inhibited.
FIG. 7 shows an expression vector for active HAC1 of S. cerevisiae (YEp351GAP-II-aHAC1), an expression vector for the human RRBP1 gene (YEp351GAP-II-p180), an expression vector for the human antibody gene (YEp352 GAP-II-alfHc/alfLc), and a coexpression vector for the activated HAC1 gene and the RRBP1 gene (YEp351GAP-II-aHAC1/p180).
FIG. 8A shows a chart showing the results of a comparison of the abilities for antibody secretory production of antibody-producing yeast strains into which an expression vector for the activated HAC1 gene of S. cerevisiae and an expression vector for the human RRBP1 gene have been introduced. FIG. 8B shows a chart showing the results of a comparison of the abilities for antibody secretory production of antibody-producing yeast strains into which an expression vector for the activated HAC1 gene of S. cerevisiae and an expression vector for the human RRBP1 gene have been introduced under conditions in which O-sugar chain formation is inhibited. FIG. 8C shows a chart showing the results of a comparison of the abilities for antibody secretory production (absolute values) of antibody-producing yeast strains into which an expression vector for the activated HAC1 gene of S. cerevisiae and an expression vector for the human RRBP1 gene have been introduced with or without the addition of a PMT inhibitor.
FIG. 9 shows the results of Western analysis of antibody production in antibody-producing strains into which a synthetic antibody gene, in which codons have been substituted, has been introduced.
FIG. 10 shows the results of Western analysis of culture supernatants of antibody-producing strains into which the activated HAC1 genes derived from yeast strains have been introduced.
FIG. 11 shows the amount of antibody secretory production from antibody-producing strains into which the activated HAC1 genes derived from yeast strains have been introduced.
FIG. 12 shows the results of Western analysis of culture supernatants of antibody-producing strains with the insertionally inactivated PMT1 gene- or PMT2 gene and those of antibody-producing strains into which the activated HAC1 genes have been introduced.
FIG. 13 shows the amount of antibody secretory production from antibody-producing strains with the insertionally inactivated PMT1 gene- or PMT2 gene and those of antibody-producing strains into which the activated HAC1 genes have been introduced.
FIG. 14 shows the results of Western analysis of culture supernatants of antibody-producing strains with the insertionally inactivated PMT4 gene and those of antibody-producing strains into which the activated HAC1 genes have been introduced.
FIG. 15 shows the amount of antibody secretory production from antibody-producing strains with the insertionally inactivated PMT4 gene and antibody-producing strains into which the activated HAC1 genes have been introduced.
FIG. 16A shows the results of Western analysis of culture supernatants of the PMT5 gene- or PMT6 gene-deficient antibody-producing strains. FIG. 16B shows the amount of antibody secretory production from the PMT5 gene- or PMT6 gene-deficient antibody-producing strains.
FIG. 17 shows the results of Western analysis of culture supernatants obtained by culturing antibody-producing strains with the insertionally inactivated PMT2 or PMT4 gene and with the activated HAC1 gene introduced therein with the addition of a PMT inhibitor (1c).
FIG. 18A shows the amount of antibody secretory production in culture supernatants obtained by culturing antibody-producing strains with the insertionally inactivated PMT2 gene and with the activated HAC1 gene introduced therein with the addition of a PMT inhibitor (1c). FIG. 18B shows the amount of antibody secretory production in a culture supernatant obtained by culturing antibody-producing strains with the insertionally inactivated PMT4 gene and with the activated HAC1 gene introduced therein with the addition of a PMT inhibitor (1c).
FIG. 19 shows the results of Western analysis of culture supernatants, which have been cultured with the addition of a variety of PMT inhibitors (rhodanine-3-acetic acid derivatives).
FIG. 20 shows the amount of antibody secretory production in culture supernatants, which have been cultured with the addition of a variety of PMT inhibitors (rhodanine-3-acetic acid derivatives).



Hereafter, the present invention is described in detail. This patent application claims priority from Japanese Patent Application No. 2006-136993 filed on May 16, 2006, and includes part or all of the contents as disclosed in the description thereof.
The present invention is composed of the following two constituents. Specifically, a combination of (A) introduction of a gene associated with high-level secretory production of a protein and (B) inhibition of O-sugar chain addition inherent to yeast (and mold) can produce synergistic effects regarding high-level secretory production of a protein.
Hereafter, the present invention is described in detail.
The present invention provides a gene used for high-level secretory production of a protein, an expression vector comprising such gene, a transformed host cell into which such expression vector is introduced, and a method for producing a protein using such transformed host cell.
1. Gene Used for High-Level Secretory Production of a Protein
(1) HAC1 Gene
In the present invention, a gene used for high-level secretory production of a protein is the HAC1 gene. The HAC1 gene is present as an inactive HAC1 gene on the genome; however, mRNA transcribed by the HAC1 gene upon endoplasmic reticulum stress application is subjected to splicing by Ire1p and converted into mRNA encoding a transcription factor, the HAC1 protein (Hac1p). The unfolded protein response (UPR) is then activated by the translated Hac1p. In the present invention, the activated HAC1 gene is defined as cDNA encoding the HAC1 protein (Hac1p) (i.e., complementary to mRNA).
Accordingly, it is preferable that the activated HAC1 gene be used in order to further improve the effects of the present invention. A given degree of effects can also be attained via introduction of the inactivated HAC1 gene.
Hereafter, the activated HAC1 gene encoding Hac1p that actually causes UPR to function is described. HAC1p is constituted by, from the N terminus, a DNA-binding domain that is highly conserved in organisms, the leucine zipper region, and an unknown active region in which mRNA is spliced and newly added by Ire1p at the C terminus.
The activated HAC1 gene used in the present invention is not particularly limited, provided that such gene encodes the activated HAC1 protein. Examples include DNA encoding a protein consisting of the amino acid sequence as shown in SEQ ID NO: 23 derived from Ogataea minuta (O. minuta), which was newly acquired in the present invention, and DNA encoding a protein consisting of the amino acid sequence derived from Pichia pastoris (P. pastoris) as shown in SEQ ID NO: 70. A functionally equivalent DNA homologous thereto may be employed.
The term “homologous DNAs” refers to, for example, a gene encoding a protein which consists of an amino acid sequence having at least 70% homology to the amino acid sequence as shown in SEQ ID NO: 23 or 70 and has the function of activating the unfolded protein response (UPR), a gene encoding a protein which consists of an amino acid sequence derived from the amino acid sequence as shown in SEQ ID NO: 23 or 70 by deletion, substitution, and/or addition of one or several amino acids and has the function of activating UPR, and a gene which hybridizes under stringent conditions to a DNA consisting of the nucleotide sequence as shown in SEQ ID NO: 22 or 69 or a complementary nucleotide sequence thereof and encodes a protein having the function of activating UPR.
The term “an amino acid sequence having at least 70% homology to the amino acid sequence as shown in SEQ ID NO: 23 or 70” refers to an amino acid sequence having preferably at least 80%, more preferably at least 90%, and most preferably at least 95% homology. Protein homology search can be carried out with the use of, for example, the DNA Databank of Japan (DDBJ) via FASTA, BLAST, or other programs.
The number indicated by the term “several” in the aforementioned “one or several amino acids in the amino acid sequence as shown in SEQ ID NO: 23 or 70” is not particularly limited. For example, the term “several” approximately refers to 20 or fewer, preferably 10 or fewer, more preferably 7 or fewer, and further preferably 5 or fewer.
Under the aforementioned “stringent conditions,” a so-called specific hybrid is formed, but a non-specific hybrid is not formed. Under such conditions, for example, complementary strands of highly homologous DNA, i.e., DNA consisting of a nucleotide sequence having at least 80%, preferably at least 90%, and more preferably at least 95% homology to the nucleotide sequence as shown in SEQ ID NO: 22 or 69 undergo hybridization, but complementary strands of DNA having lower homology levels would not undergo hybridization. More specifically, the sodium concentration is 150 to 900 mM, and preferably 600 to 900 mM, and the temperature is 60° C. to 68° C., and preferably 65° C.
The mutation mentioned above, such as deletion, substitution, and/or addition, can be introduced via a technique known in the art, such as the Kunkel method or the Gapped duplex method, or a technique in accordance therewith. For example, mutagenesis kits utilizing site-directed mutagenesis, such as a Mutant-K (Takara Bio), Mutant-G (Takara Bio), or LA PCR in vitro Mutagenesis series kit (Takara Bio), can be used.
The term “function of activating UPR” refers to the function of activating the defense reactions of endoplasmic reticulum (ER) against accumulation of unfolded proteins (e.g., inhibition of transcription, acceleration of folding induced by molecular chaperons, degradation of denatured protein, or cell death caused by apoptosis). The function of activating UPR is substantially equivalent to the function of a gene encoding a protein consisting of the amino acid sequence as shown in SEQ ID NO: 23 or 70.
The activated HAC1 gene may be a gene of O. minuta, P. pastoris, or another methanol-assimilating yeast. Examples thereof include genes encoding activated HAC1 proteins derived from Hansenulla polymorpha (Pichia angusta), Pichia methanolica, and Candida boidinii. Also, it may be an activated HAC1 gene derived from another organism species, such as other types of yeast or mold. Examples thereof include a gene (YFL031W, GenBank accession number: DNA spliced from D26506 by Ire1p) encoding the activated HAC1 protein (GenBank accession number: NP—116622) derived from Saccharomyces cerevisiae (S. cerevisiae), a gene (GenBank accession number: AJ413272) encoding the activated HAC1 protein (GenBank accession number: CAC88374) derived from Trichoderma reesei, and a gene (GenBank accession number: AJ413273) encoding the active HacA protein (GenBank accession number: CAC88375) derived from Aspergillus nidulans. 
The nucleotide sequences of genes encoding the activated HAC1 proteins derived from S. cerevisiae, Trichoderma reesei, and Aspergillus nidulans are shown in SEQ ID NOs: 39, 41, and 43, and the corresponding amino acid sequences are shown in SEQ ID NOs: 40, 42, and 44.
The activated HAC1 genes derived from Ogataea minuta and Pichia pastoris isolated herein were the first genes isolated from yeast strains other than Saccharomyces cerevisiae. This strongly suggests the presence of the gene of interest generally in yeast strains, such as methanol-assimilating yeast strains. Accordingly, these genes are within the scope of the activated HAC1 genes used in the present invention.
Further, a transcription factor that activates UPR may be used as an alternative to the aforementioned activated HAC1 gene. An example is a gene that is activated upon splicing, by Ire1p, from the XBP-1 gene (e.g., a human-derived gene with GenBank accession number: NM—005080), which is an HAC1 homolog derived from animal cells or other species. Artificial activation by Ire1, which also activates HAC1 (XBP-1), also corresponds to activation of UPR. Accordingly, it is considered to be equivalent to introduction of the activated HAC1 gene. Further, forced expression of the non-activated HAC1 gene is considered to yield equivalent effects to the case of introduction of activated HAC1 as described above.
The activated HAC1 gene can be obtained by any method, provided that UPR is induced. For example, mRNA may be obtained from a cell in which genes encoding proteins that are difficult to fold are expressed at high level or cells that are treated with a sugar chain modification inhibitor, such as tunicamycin, a redox agent, such as DTT or hydrogen peroxide, or a UPR inducer, following which cDNA is synthesized therefrom. Also, mRNA may be obtained from a sequence that is already disclosed by synthesizing a part of or the full length thereof with the use of a DNA synthesizer.
(2) RRBP1 Gene
In the present invention, an example of another gene used for high-level secretory production of a protein is the RRBP1 gene. The RRBP1 gene is a gene encoding a protein referred to as the ribosome-binding protein 1, and it is also referred to as the hES, ES130, ES/130, or DKFZp586A1420 gene. The mammalian RRBP1 gene is composed of the N-terminal transmembrane region, a subsequent region that is rich in basic amino acids, 54 repeats of a sequence comprising 10 amino acid residues, and the C-terminal region.
The RRBP1 gene used in the present invention is not particularly limited, provided that it encodes the ribosome-binding protein 1. Examples include the human-derived RRBP1 gene (encoding the KIAA 1398 protein; GenBank accession number: AB037819) and the dog-derived RRBP1 gene (encoding the ribosome receptor p180; GenBank accession number: X87224). As long as it is functionally equivalent to the aforementioned genes, a homologous gene thereof may be used. The nucleotide sequences of the human-derived RRBP1 gene and the dog-derived RRBP1 gene are shown in SEQ ID NOs: 45 and 47, and the corresponding amino acid sequences are shown in SEQ ID NOs: 46 and 48.
Examples of homologous genes include: a gene encoding a protein which consists of an amino acid sequence having at least 70% homology with the amino acid sequence of human- or dog-derived RRBP1 and has ribosome-binding activity; a gene encoding a protein which consists of an amino acid sequence derived from the amino acid sequence of human- or dog-derived RRBP1 by deletion, substitution, and/or addition of one or several amino acids and has ribosome-binding activity; and a gene which hybridizes under stringent conditions to a gene consisting of a nucleotide sequence of the human- or dog-derived RRBP1 gene or a complementary nucleotide sequence thereof and encodes a protein having ribosome-binding activity. The degree of homology, stringent conditions, and a method of mutagenesis are as described above.
Specific examples of such homologous genes include RRBP1 genes derived from mice (accession Nos: XM—622097, XM—91338, and XM—991888), a rat (accession No. XM—230637), a Xenopus (accession No: NM—001005671), and a zebra fish (zebra danio) (accession No: NM—199431).
The RRBP1 gene may also be obtained by a generally known technique. For example, mRNA may be prepared from a cell in which the RRBP1 gene is expressed, and cDNA may further be synthesized.
In the present invention, the aforementioned gene used for high-level secretory production of a protein and a gene encoding a foreign protein that is the target of high-level secretory production described below (hereafter these genes are referred to as “target genes”) can be obtained by a general technique of preparing mRNA and synthesizing cDNA using reverse transcriptase. As an example of the aforementioned general technique, a cDNA library derived from a cell or tissue in which the target gene is expressed is subjected to screening with the use of a DNA probe synthesized from a fragment of the target gene, so as to isolate the gene of interest. mRNA can be prepared by a technique generally used in the art. For example, the aforementioned cell or tissue may be treated with a guanidine reagent or a phenol reagent to obtain total RNA, following which poly (A)+ RNA (mRNA) is then obtained via the affinity column method using oligo (dT) cellulose columns or poly U-sepharose using sepharose 2B as a carrier or a batch technique. Further, poly (A)+ RNA may be fractionated via sucrose density gradient centrifugation or via other means. Subsequently, the obtained mRNA is used as a template to synthesize single-stranded cDNA using oligo dT primers and reverse transcriptase, and double-stranded cDNA is synthesized from the single-stranded cDNA using DNA synthetase I, DNA ligase, RnaseH, and the like. The synthesized double-stranded cDNA is blunt-ended using T4 DNA synthetase, subjected to ligation of an adaptor (e.g., an EcoRI adaptor), phosphorylation, or the like, incorporated into a λ phage, such as λgt11, and then packaged in vitro to prepare a cDNA library. In addition to a λ phage, plasmid vectors may be used to prepare cDNA library. Thereafter, a strain having DNA of interest (i.e., a positive clone) may be selected from the cDNA library.
When the target gene is isolated from genomic DNA or when a fragment containing a promoter region and a terminator region is isolated, genomic DNA is extracted from a cell strain of a source organism, and the target gene is selected in accordance with a common technique (Molecular Cloning, 1989; Methods in enzymology 194, 1991). Genomic DNA can be extracted by the method of Cryer et al. (Methods in Cell Biology, 12, 39-44, 1975) or the method of P. Philippsen et al. (Methods Enzymol., 194, 169-182, 1991), for example. When the source is a yeast strain, for example, a yeast protoplast is prepared, and the yeast protoplast is then subjected to a conventional technique, such as known DNA extraction techniques, alcohol precipitation techniques after removal of cell residues at a high salt concentration, or alcohol precipitation techniques after phenol or chloroform extraction.
The target gene can be obtained by, for example, PCR (PCR Technology, Henry A. Erlich, Stockton Press, 1989). When amplifying the target gene via PCR, a synthesized 20mer to 30mer single-stranded DNA is used as a primer, and genomic DNA is used as a template. The nucleotide sequence of the amplified gene is confirmed and then used.
A fragment containing a target gene whose sequence is unknown can be obtained by (a) preparing a gene library by a conventional technique and (b) selecting a clone of interest from the resulting gene library to be amplified. A gene library can be prepared by obtaining chromosome DNA from a cell line of a source organism via a conventional technique, partially digesting the chromosome DNA with adequate restriction enzymes for fragmentation, ligating the resulting fragment to an adequate vector, and then introducing the vector into an adequate host cell. Alternatively, mRNA may be extracted from a cell, cDNA may be synthesized therefrom, the synthesized cDNA may be ligated to an adequate vector, and the vector may be introduced into an adequate host cell, so that a gene library can be prepared. In such a case, a plasmid that is known as a conventional vector for gene library preparation can be used, and phages, cosmids, or other vectors can be extensively used. A host cell that is subjected to transformation or transduction may be selected in accordance with vector type.
Clones that carry target gene fragments are selected from the above gene library via colony hybridization, plaque hybridization, or other means involving the use of label probes containing sequences specific to the target genes.
Also, the target genes can be subjected to chemical total synthesis. For example, two pairs of complementary oligonucleotides are prepared and then annealed, several annealed DNA strands are ligated with the aid of DNA ligase, or several partially complementary oligonucleotides are prepared and gaps are filled by PCR. Thus, genes can be synthesized.
DNA sequences of genes can be determined by a conventional technique, such as the dideoxy method (Sanger et al., Proc. Natl. Acad. Sci., U.S.A., 74, 5463-5467, 1977). Further, the above DNA nucleotide sequences can be easily determined with the use of a commercially available sequencing kit or the like.
2. Expression Vector
The present invention provides a vector comprising the activated HAC1 gene or the RRBP1 gene or a vector comprising both the activated HAC1 gene and the RRBP1 gene. In order to express the activated HAC1 gene and the RRBP1 gene in host cells, a vector comprising either the activated HAC1 gene or the RRBP1 gene may be used to carry out transformation. Alternatively, a vector comprising both such genes may be used to carry out transformation. Also, such expression vector may comprise a gene encoding a foreign protein. Alternatively, an expression vector comprising a gene encoding a foreign protein may be prepared separately. In such a case, vectors are cotransfected into a host cell.
A gene encoding a foreign protein is not particularly limited. Examples include: various enzyme genes, such as the α-amylase gene and the α-galactosidase gene; various interferon genes that are pharmaceutically useful and physiologically active proteins, such as interferon α and interferon γ; various interleukin genes, such as IL1 and IL2; various cytokine genes, such as the erythropoietin (EPO) gene and the granulocyte colony-stimulating factor (G-CSF) gene; and growth factor genes. These genes may be obtained via any means.
The present invention is particularly effective on a protein that is highly hydrophobic and a protein whose secretory production is insufficient due to composite formation. Thus, the aforementioned foreign protein includes a multimeric protein, such as an antibody or its functional fragment, i.e., a heteromultimer.
An expression regulation region may be adequately added to the activated HAC1 gene, the RRBP1 gene, or a gene encoding a foreign protein to constitute an expression vector as a protein expression unit. A protein expression unit comprises, in the direction of a reading frame of transcription, at least a promoter region, the above gene, and a transcription terminator region. A promoter that can be used herein may be an inducible expression promoter or constitutive expression promoter. Examples of inducible expression promoters include promoters involved in methanol metabolism of methanol-assimilating yeast, such as alcohol oxidase (AOX) gene promoters, dihydroxyacetone synthase (DAS) gene promoters, and formate dehydrogenase (FDH) promoters. An example of another inducible promoter that can be used is a copper-inducible (CUP) promoter. Examples of constitutive expression promoters include promoters of the glyceraldehyde-3-phosphate dehydrogenase (TDH, GAP) gene, the phosphoglycerokinase (PGK) gene, the phosphotriose isomerase (TPI) gene, the enolase (ENO) gene, the actin (ACT) gene, the cytochrome c (CYC) gene, the trehalose synthase (TPS) gene, and the alcohol dehydrogenase (ADH) gene. Also, a transcription terminator may be a sequence having activity of terminating transcription from a promoter. It may be a sequence of the same or a different gene of the promoter.
In order to realize high-level secretory production of foreign proteins, use of a potent promoter is necessary. When production of a protein that is less likely to fold or less likely to be secreted is attempted with the use of a highly active promoter, hyposecretion may disadvantageously occur. Such hyposecretion occurs due to the following reasons. That is, protein production exceeds the capacity of the ribosome where translation is performed and the endoplasmic reticulum wherein folding and secretion are performed. This causes excessively produced proteins to be denatured, accumulated, ubiquitinated in cells, and degraded by the proteosome. Accordingly, promoters that can attain an expression level to the extent that resulting proteins would be denatured and would not undergo aggregation or the resulting proteins would not exceed the secretion capacity may be adequately selected. Alternatively, activity of the promoters may be attenuated and the promoters of interest may then be used. Molecules that form heteromultimers are likely to be affected as described above among multimeric proteins. In particular, molecules such as antibodies are heterotetramers comprising two molecules each of the heavy chain and of the light chain being aggregated. Thus, the expression level is an important factor for realizing adequate aggregation. When the expression intensity of the activated HAC1 gene is excessively strong, excessive stress is applied to a cell, and this may disadvantageously inhibit growth. Thus, adjustment and optimization of promoter activity are required as described above.
The expression vector of the present invention can comprise a selection marker for selecting a transformant. For examples, expression vectors for yeast can comprise auxotrophic marker genes selected from among His1, His2, His3, His4, His5, His6, Leu2, Alg1, Alg2, Alg3, Trp1, Lys2, Ade1, Ade2, Ura3, and Ura5 genes.
As selection markers, drug-resistant markers that impart resistance to drugs such as cerulenin, aureobasidin, Zeocin, canavanine, cycloheximide, hygromycin, blasticidin, tetracycline, kanamycin, ampicillin, tetracycline, and neomycin can be used, in addition to the aforementioned auxotrophic markers. Thus, transformants can be selected. Also, genes that impart solvent resistance to ethanol, osmotic resistance to glycerol or salt, metal ion resistance to copper, and the like may be used as markers, so that transformants can be selected.
3. Transformed Host Cell
The transformed host cell of the present invention comprises the gene described in 1. above or the expression vector described in 2. above introduced therein.
An example of a host cell to be transformed is an eucaryotic cell, and preferably a yeast strain. Examples of yeast strains include methanol-assimilating yeast strains, such as Ogataea minuta, Pichia pastoris, Hansenulla polymorpha (Pichia angusta), and Candida boidinii and yeast strains, such as Saccharomyces cerevisiae, Kluyveromyces lactis, Yarowia lipolytica, and Shizosaccharomyces pombe. More specifically, the Ogataea minuta YK3 strain (Δoch1Δpep4Δprb1Δyps1Δura3Δade1) can be used as the Ogataea minuta strain, and the Saccharomyces cerevisiae BY4741 strain (MATaΔhis3Δleu2Δmet15Δura3) can be used as the Saccharomyces cerevisiae strain, although the yeast strains are not limited thereto.
Further, the present invention is intended to obtain a host cell in which the ER, which is essential for secretion, is enhanced. Accordingly, the present invention is applicable to animal cells or other cells.
In the present invention, an expression vector can be introduced into a host cell by any method, provided that an introduced gene is stably present and adequately expressed in a host. Examples of such methods that are generally employed include the calcium phosphate method (Ito et al., Agric. Biol. Chem., 48, 341, 1984), electroporation (Becker, D. M. et al., 1990; Methods. Enzymol., 194, 182-187), use of spheroplasts (Creggh et al., Mol. Cell. Biol., 5, 3376, 1985), the lithium acetate method (Itoh, H., 1983; J. Bacteriol. 153, 163-168), and lipofection.
4. Method for Producing Protein
In the present invention, proteins can be produced by culturing the transformed host cells via a conventional technique and sampling the proteins from the culture product, followed by purification. The term “culture product” used herein refers to culture cells, cultured strains, or disrupted cells or bacteria, in addition to a culture supernatant.
The transformed host cell can be cultured in a medium in accordance with a conventional method used for culture of the host cell.
When the transformed host cell is a microorganism, such as yeast, either a natural or synthetic medium may be used as a medium for culture, provided that it contains carbon sources, nitrogen sources, and inorganic salts assimilable by the microorganism and is capable of efficient culture of the transformant. Any carbon sources assimilable by the microorganism may be used, and examples thereof include: carbohydrates such as glucose, fructose, sucrose, and starch; organic acids such as acetic acid and propionic acid; and alcohols such as ethanol and propanol. Examples of nitrogen sources include: ammonia; ammonium salts of inorganic or organic acids such as ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate; other nitrogen-containing compounds; peptone; meat extracts; and corn steep liquor. Examples of inorganic salts include: monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, iron(I) sulfate, manganese sulfate, copper sulfate, and calcium carbonate. In accordance with the type of selection marker, an antibiotic agent, such as aureobasidin, ampicillin, or tetracycline, may be adequately added to a medium. Alternatively, an amino acid that can be supplied by a gene complementing auxotrophy (e.g., Leu, Ura, or Trp) may be removed.
When culturing the transformed host cell, in the case of yeast, for example, the pH level of the medium is preferably adjusted to 4 to 7. The culture temperature is between 15° C. and 32° C., and preferably around 28° C. When a protein having a complicated steric structure as an antibody is expressed, culture may be preferably carried out at a low temperature, in order to more effectively fold such a protein in the cell. The culture duration is generally about 24 to 1,000 hours, and culture can be carried out via batch culture, such as static, shake, agitation, or aeration culture, or via continuous culture.
An expression product of a gene of a foreign protein from the culture product (i.e., a culture solution or cultured cells) can be confirmed via SDS-PAGE, Western blotting, ELISA, or the like.
The produced proteins may be isolated and purified via conventional techniques for protein isolation and purification. When target proteins are produced in the bacteria or cells after culture, the bacteria or cells may be pulverized using, for example, an ultrasonic pulverizer, a French press, a Manton-Gaulin homogenizer, Dinomil, or the like, to obtain target proteins. When the target proteins are produced outside the bacteria or cells, the culture solution is used as it is, or the bacteria or cells are removed via centrifugation or the like. Thereafter, the target proteins are collected via extraction using an organic solvent, subjected to various chromatography techniques (e.g., hydrophobic, reversed-phase, affinity, or ion-exchange chromatography), gel filtration using molecular sieves, electrophoresis using polyacrylamide gel, or the like, according to need. These techniques may be employed solely or in combinations of two or more. Thus, the target proteins may be isolated and purified.
The above culture and purification techniques are examples, and methods are not limited thereto. The amino acid sequence of the purified gene product can be confirmed by a conventional method of amino acid analysis, such as automated amino acid sequencing via the Edman degradation technique.
5. Method for Inhibiting O-Sugar Chain (or Method for Inhibiting PMT Activity)
In the present invention, when yeast is used as a host cell, the aforementioned culture is preferably carried out under conditions in which protein O-mannosyltransferase (PMT) activity is inhibited.
An O-sugar chain is formed in a mammalian cell upon the addition of GalNAc by peptide O-GalNAc transferase, which is present mainly in the Golgi apparatus. Such sugar chain addition takes place after protein folding. In contrast, O-sugar chain formation in yeast and mold cells is initiated upon the addition of mannose to a serine or threonine residue of the protein by a protein-O-mannosyltransferase (PMT) encoded by the PMT gene. Such addition is referred to as PMT activity. The addition of mannose takes place in parallel with protein folding in the endoplasmic reticulum (ER) in the cell. Thus, an unnecessary sugar chain may be disadvantageously added to a site at which such addition would not take place in the case of expression of mammalian proteins. Consequently, such unnecessary modification would cause insufficient formation of aggregates and lower the activity.
By performing culture under conditions in which protein O-mannosyltransferase (PMT) activity is inhibited, accordingly, formation of an unnecessary 0 sugar chain can be inhibited. This also accelerates protein aggregation and enables maintenance of indigenous physical properties and activity of proteins. In the present invention, effects of high-level secretory production of proteins via introduction of the activated HAC1 gene and/or the RRBP gene can further produce synergistic effects by regulating the O-sugar chain formation enhanced by URP via inhibition of PMT activity.
Addition of an O-sugar chain peculiar to yeast or a mold can be inhibited by, for example, the two methods described below. These methods can be performed in combination.
(1) Culture and production are carried out under conditions in which PMT activity that undergoes addition of an O-sugar chain peculiar to yeast or a mold is inhibited.
(2) Cells in which PMT activity that undergoes addition of an O-sugar chain peculiar to yeast or a mold is inhibited are used.
The protein O-mannosyltransferase (PMT) activity of (1) above can be inhibited with the addition of an inhibitor of PMT activity (i.e., a PMT inhibitor) to the medium, for example. An example of an inhibitor of PMT activity that can be used is the rhodanine-3-acetic acid derivative (Bioorganic & Medicinal Chemistry Letters 14, pp. 3975-3978, 2004). Specific examples of the rhodanine-3-acetic acid derivative include 5-[[3,4-(1-phenylmethoxy)phenyl]methylene]-4-oxo-2-thioxo-3-thiazolidineacetic acid (compound (1c) described in Bioorganic & Medicinal Chemistry Letters, Vol. 14, p. 3975, 2004) and {(5Z)-4-oxo-5-[3-(1-phenylethoxy)-4-(2-phenylethoxy)benzylidene]-2-thioxo-1,3-thiazolidin-3-yl}acetic acid (compound (5a) described in Bioorganic & Medicinal Chemistry Letters, Vol. 14, p. 3975, 2004). Such inhibitor of PMT activity (the rhodanine-3-acetic acid derivative) was first examined as an antibacterial agent, and it was not examined for the purpose of improving protein quality or productivity. The effects thereof were first discovered in the present invention. PMT is important for generation of mannoproteins that constitute the yeast strain wall. Excessively lowered PMT activity would adversely affect the growth of yeast. When inducible expression systems are used, accordingly, the addition of an inhibitor of PMT activity at the time of expression of genes of foreign proteins, following cell growth, would be more effective. Thus, high-quality target proteins in which O-sugar chain modification is inhibited can be produced at the maximum level.
The protein O-mannosyltransferase (PMT) activity described in (2) above can be inhibited by disrupting the PMT gene or inhibiting expression of such gene. In S. cerevisiae, PMT is encoded by at least 6 genes; i.e., the PMT1 gene (GenBank: L19169), the PMT2 gene (GenBank: L05146), the PMT3 gene (GenBank: X83797), the PMT4 gene (GenBank: X83798), the PMT5 gene (GenBank: X95644), and the PMT6 gene (GenBank: Z72984), and these genes independently form a homodimer (PMT4p) or a heterodimer (PMT1p/PMT2p) and exhibit activity. It is known that acting PMT varies in accordance with a glycoprotein. In the present invention, PMT proteins were found to have selectivity with regard to the addition of an O-sugar chain to an antibody. Specifically, the effects of inhibition of O-sugar chain addition were not found in the PMT5 or PMT6 gene-deficient strain, as described in the examples.
Thus, PMT is an important gene for the growth of yeast. When activity, such as disruption of the PMT gene, is eliminated or extremely lowered, the cell wall becomes fragile. Thus, the use of a PMT gene-deficient strain requires attention. Disruption of PMT genes as disclosed in WO 2002/046437 is not always effective. It may sometimes adversely affect the growth of a foreign protein due to growth inhibition, and inhibition of disruption or expression of PMT genes having optimal PMT activity, which can minimize O-sugar chain addition and modification to the target protein, is desired. Examples of methods for inhibiting PMT genes include a method involving the use of antisense RNA or RNAi and a method of attenuating a promoter. In the present invention, a method in which a DNA fragment is inserted into the PMT structural gene portion and the promoter region to cleave the gene (hereafter, such method may be referred to as “insertional inactivation of genes,” and a plasmid vector for insertional inactivation of genes is referred to as a “insertional inactivation vector”) is demonstrated as an example of a means for attenuating a promoter. Also, a method wherein a PMT gene fragment that does not have PMT activity but is generated as a protein or a gene whose activity-related amino acid residue has been mutated is introduced to inhibit PMT activity (i.e., a dominant-negative method) can also be employed.
BEST MODES FOR CARRYING OUT THE INVENTION
Hereafter, the present invention is described in detail with reference to the examples, although the technical scope of the present invention is not limited to the examples. Plasmids, restriction enzymes, DNA modifying enzymes, and the like that are used in the examples of the present invention are commercially available products, and these products can be used in accordance with conventional techniques. Also, procedures of DNA cloning, nucleotide sequencing, host cell transformation, culture of transformed host cells, sampling and purification of enzymes from culture products, and the like are well-known in the art or can be learned through existing publications.
EXAMPLE 1
Construction of Foreign Gene-Expressing Plasmid
(1) Construction of Foreign Gene-Expressing Tandem Vector Comprising the AOX1 Gene Promoter Having the G418-Resistant Gene as the Marker, the Terminator Cassette and the GAP Gene Promoter, and the Terminator Cassette
pOMex3G and pOMexGP1U disclosed in WO 2003/091431 were used as materials. pOMex3G was cleaved with XbaI and blunt-ended, followed by introduction of the SpeI linker. The resulting vector was designated as pOMex3GXS. Separately, pOMexGP1U was cleaved with EcoT22I and blunt-ended, followed by introduction of the ApaI linker. The resulting vector was designated as pOMexGP1UTA. pOMexGP1UTA was digested with HindIII and KpnI and blunt-ended. Thereafter, the isolated fragment of about 2.0 kb containing a GAP promoter and a terminator was digested with ApaI and then introduced into the blunt-ended pOMex3GXS. The resulting vector was designated as pOMexGAT-G. pOMexGAT-G is a tandem vector that comprises the SpeI-BamHI site within the AOX1 expression cassette and the SalI-ApaI site within the GAP expression cassette.
(2) Construction of a Foreign Gene-Expressing Vector with the Use of a Gap Gene Promoter and a Terminator Using the ADE1 Gene as a Selection Marker
pOMex4A disclosed in WO 2003/091431 was used as a material. The aforementioned pOMexGP1U was treated with EcoT22I and blunt-ended, followed by introduction of the BamHI linker. The resulting vector was designated as pOMexGP2U. pOMexGP2U was treated with SalI and blunt-ended, followed by introduction of the SpeI linker. The resulting vector was designated as pOMexGP3U. pOMexGP3U was digested with HindIII and KpnI, and a fragment of approximately 2.0 kb containing the GAP expression cassette was isolated. The resulting fragment was ligated to a fragment of approximately 5.0 kb comprising the ADE1 marker isolated by treating pOMex4A with HindIII-KpnI. The resulting vector was designated as pOMexGP1A. pOMexGP1A is a foreign gene-expressing vector that comprises the SpeI-BamHI site within the GAP expression cassette.
(3) Construction of a Foreign Gene-Expressing Vector with a Phosphoglycerine Kinase (PGK1) Promoter and a Terminator Using a Hygromycin B-Resistant Gene as a Selection Marker
The PGK1 gene encoding phosphoglycerine kinase was obtained from the Ogataea minuta IFO10746 strain, and the nucleotide sequence thereof was determined.
(3-1) Preparation of Probes
DNA degenerate primers comprising nucleotide sequences corresponding to the conserved amino acid sequences, i.e., RVDFNVPLD (SEQ ID NO: 123) and EGKELPGVA (SEQ ID NO: 124), derived from Saccharomyces cerevisiae (Gen Bank accession number: P00560) and Candida maltosa (GenBank accession number: P41757) were synthesized in the following manner.










PPG5:









(SEQ ID NO: 1)











5′-GN GTN GAY TTY AAY GTN CCN TTR GA-3′




 



PPG3:








(SEQ ID NO: 2)











5′-GY NAC DCC NGG YAA YTC YTT DCC YTC-3′







The PPG5 primer (SEQ ID NO: 1) corresponds to the amino acid sequence, RVDFNVPLD (SEQ ID NO: 123), and the PPG3 primer (SEQ ID NO: 2) is a sequence of a complementary strand of a nucleotide sequence corresponding to the amino acid sequence, EGKELPGVA (SEQ ID NO: 124). Chromosome DNA of the O. minuta IFO10746 strain was used as a template, PCR was carried out using PPG5 and PPG3 primers at 94° C. for 30 seconds, 50° C. for 1 minute, and 72° C. for 1 minute, and this cycle was repeated 25 times. The amplified DNA fragment (approximately 1.2 kb) was recovered and cloned using the TOPO TA Cloning Kit. Plasmid DNA was isolated from the resulting clone, and the nucleotide sequence was determined. Thus, a clone having a nucleotide sequence encoding an amino acid sequence having high homology to the amino acid sequence of the PGK1 gene derived from S. cerevisiae and C. maltosa in the plasmid-inserted DNA fragment was selected. The 1.2-kb DNA-inserted fragment was recovered after the plasmid was cleaved with EcoRI, followed by agarose gel electrophoresis.
(3-2) Preparation and Screening of Library
Chromosome DNA of the O. minuta IFO10746 strain was cleaved with various restriction enzymes and 0.8% agarose gel electrophoresis was performed. The separated DNA was transferred on the Hybond N+ nylon membrane (Amersham). The DNA fragment obtained in (1-3-1) above was labeled with the use of the AlkPhos DIRECT (Amersham), followed by Southern hybridization. Hybridization was carried out in accordance with a conventional technique (Molecular cloning 2nd edn., ed. Sambrook, J., et al., Cold Spring Harbor Laboratory U.S.A., 1989). As a result, the PGK1 gene was considered to be present in a BamHI fragment of approximately 9.0 kb. In order to clone the DNA fragment, genome library was prepared. Chromosome DNA of O. minuta was cleaved with BamHI and subjected to agarose electrophoresis, and a DNA fragment of approximately 9.0 kb was recovered from the gel. The recovered DNA fragment was ligated to the BamHI-cleaved pUC118 and transformed into the E. coli DH5α strain in accordance with the method of Hanahan (Gene, 10, 63, 1980) to prepare library. Approximately 4,000 clones were screened via colony hybridization using the aforementioned DNA fragments as probes. From among the obtained positive clones, the pOMPGK1 plasmids carrying PGK1 genes were selected.
(3-3) Nucleotide Sequencing
The nucleotide sequence in the BamHI region in the pOMPGK1 plasmid was determined by the primer walking method, and the determined sequence was found to have the nucleotide sequence as shown in SEQ ID NO: 3. The nucleotide sequence as shown in SEQ ID NO: 3 comprises an open reading frame comprising 1,254 base pairs from nucleotides 4,766 to 6,016. Homology between the amino acid sequence as shown in SEQ ID NO: 4 that is deduced based on the open reading frame and phosphoglycerine kinases derived from Saccharomyces cerevisiae and Candida maltosa was inspected. As a result, the former homology was found to be 74%, and the latter homology was found to be 81%.
(3-4) Construction of Foreign Gene-Expressing Cassette Using PGK1 Gene Promoter and Terminator
An expression cassette that introduces a foreign gene between a fragment containing the PGK1 gene promoter and a fragment containing a terminator of the O. minuta was prepared. In order to introduce the SpeI, BglII, and BamHI sites between the PGK1 gene promoter and the terminator, the following primers were synthesized.








OPGK-P-F:









(SEQ ID NO: 5)









5′-AAGCTTGACAATGTAGGAGATCATAAACACATCGTGCGCGTC-3′



 


OPGK-P-R:








(SEQ ID NO: 6)









5′-GGATCCAGATCTCATATGACTAGTTGCTAGTTCTATGCGGCGTTAGT



 


GTTTACACTACGACAGCT-3′


 


OPGK-T-F:








(SEQ ID NO: 7)









5′-GGATCCGTGGGATTTGCGTGATCTACGTAGTGGTTATTTT-3′



 


OPGK-T-R:








(SEQ ID NO: 8)









5′-GGTACCGCAGTGAAAGGCGATGCCACCATGTGCAAGGAGTTC-3′







Using pOMPGK1 above as a template, PCR was carried out using the OPGK-P-F primer (SEQ ID NO: 5) and the OPGK-P-R primer (SEQ ID NO: 6) at 94° C. for 30 seconds, 55° C. for 1 minute, and 72° C. for 1 minute, and this cycle was repeated 20 times. Also, PCR was carried out using the OPGK-T-F primer (SEQ ID NO: 7) and the OPGK-T-R primer (SEQ ID NO: 8) at 94° C. for 30 seconds, 55° C. for 1 minute, and 72° C. for 1 minute, and this cycle was repeated 20 times. The amplified 1.5-kb and 1.0-kb DNA fragments were recovered and cloned using the TOPO TA Cloning Kit. The nucleotide sequences of the insertion DNA fragments were determined to select clones having the correct nucleotide sequences. The 1.5-kb and 1.0-kb insertion DNA fragments were isolated as the HindIII-BamHI fragment and the BamHI-KpnI fragment, respectively.
The aforementioned 1.0-kb BamHI-KpnI fragment was introduced between BamHI and KpnI of pOMex5H described in WO 2003/091431. Thereafter, the aforementioned 1.5-kb HindIII-BamHI fragment was introduced between HindIII and BamHI of the obtained plasmid. The resulting plasmid was designated as pOMexPGHy. pOMexPGHy is a foreign gene-expressing vector comprising SpeI, BglII, and BamHI sites in the PGK1 gene expression cassette.
EXAMPLE 2
Construction of Antibody Gene Expression Vector
In order to clone a secretion signal of MF alpha1 (GenBank accession number: P01149) derived from S. cerevisiae (hereafter referred to as the “aMF secretion signal”), the following primers were synthesized.









Sp-aMFs-F:




5′-ACTAGTATGAGATTTCCTTCAATTT-3′
(SEQ ID NO: 9)


 


Sl-aMFs-F:


5′-GTCGACATGAGATTTCCTTCAATTT-3′
(SEQ ID NO: 10)


 


Xb-aMFs-R:


5′-AGCTTCAGCCTCTCTTTTATCTAGAGA-3′
(SEQ ID NO: 11)






Genome DNA of S. cerevisiae obtained in the same manner as described above was used as a template to carry out PCR using the Sp-aMFs-F primer (SEQ ID NO: 9) and Xb-aMFs-R primer (SEQ ID NO: 11) at 94° C. for 30 seconds, 55° C. for 1 minute, and 72° C. for 30 seconds, and this cycle was repeated 20 times. Also, PCR was carried out using the Sl-aMFs-F primer (SEQ ID NO: 10) and the Xb-aMFs-R primer (SEQ ID NO: 11) at 94° C. for 30 seconds, 55° C. for 1 minute, and 72° C. for 30 seconds, and this cycle was repeated 20 times. The amplified DNA fragments of approximately 0.3 kb in each PCR were recovered and cloned using the TOPO TA Cloning Kit. The nucleotide sequences of the insertion DNA sequences were confirmed, and the resulting plasmids were designated as TOPOaMFsSP and TOPOaMFsSL, respectively.
The anti-TRAIL receptor antibody gene (WO 2002/094880) was used as an antibody gene. In order to introduce restriction enzyme sites into sites at both terminuses of the light-chain and heavy-chain genes, the following primers were synthesized.








Xb-KREAEA-Hc-F:









(SEQ ID NO: 12)









5′-TCTCTAGATAAAAGAGAGGCTGAAGCTCAGCTGCAGCTGCAGGAG



 


TC-3′


 


Hc-R-Bg:








(SEQ ID NO: 13)









5′-CCAGATCTGGATCCTCATTTACCCGGAGACAGGGAGAGG-3′



 


Xb-KREAEA-Lc-F:








(SEQ ID NO: 14)









5′-TCTCTAGATAAAAGAGAGGCTGAAGCTGAAATTGTGTTGACACAG



 


TC-3′


 


Lc-R-Ap:








(SEQ ID NO: 15)









5′-AAAGGGCCCTCAACACTCTCCCCTGTTGAAGCTCT-3′







Using these DNA primers, PCR was carried out to amplify a light chain via 20 cycles of 94° C. for 30 seconds, 55° C. for 1 minute, and 72° C. for 1 minute and to amplify a heavy chain via 20 cycles of 94° C. for 30 seconds, 55° C. for 1 minute, and 72° C. for 1 minute and 30 seconds, and the amplified products were cloned into pCR2.1-TOPO. The nucleotide sequences of the insertion DNA sequences were confirmed, the resulting plasmids were designated as TOPOHc-Trail and TOPOLc-Trail, respectively. An aMF secretion signal isolated from TOPOaMFsSL via digestion with SalI and XbaI and an antibody light chain isolated from TOPOLc-Trail via digestion with XbaI and ApaI were introduced into the SalI-ApaI-digested pOMexGAT-G via 3 fragment ligation. The resulting plasmid was designated as pOMexGAT-G/L. Subsequently, an aMF secretion signal isolated from TOPOaMFsSP via digestion with SpeI and XbaI and an antibody heavy chain isolated from TOPOHc-Trail via digestion with XbaI and BglII were introduced into the SpeI-BamHI-digested pOMexGAT-G/L via 3 fragment ligation. The resulting vector was designated as pOMexGAT-G/Ab (FIG. 3). pOMexGAT-G/Ab is an antibody expression vector comprising both antibody heavy chain and light chain expression units.
EXAMPLE 3
Construction of Activated HAC1 Gene Expression Vector of O. minuta 
The activated HAC1 gene was obtained by culturing cells (O. minuta YK2-3 strains) in YPD medium at 27° C. for 12 hours and adding tunicamycin to a concentration of 10 μg/ml in the medium so as to induce UPR. Culture was carried out in a tunicamycin-containing medium for an additional 12 hours. After the cells were collected, mRNA was prepared using the Yeastar RNA kit (ZYMO research).
The obtained mRNA was subjected to DNase treatment using DNase I Amplification Grade (Invitrogen). cDNA was synthesized from the mRNA using Super script III First-Strand Synthesis for RT (Invitrogen). The resulting cDNA was amplified via PCR using the HAC1-1 DNA primer (SEQ ID NO: 16) and the HAC1-12 primer (SEQ ID NO: 17) described below at 94° C. for 30 seconds, 52° C. for 30 seconds, and 72° C. for 1 minute, and this cycle was repeated 30 times. The amplified products were cloned into pCR2.1-TOPO (Invitrogen). Thereafter, the nucleotide sequences of the two types of PCR-amplified gene fragments were confirmed (SEQ ID NOs: 18 and 19).









HAC1-1:




5′-ATGACTTCCTTTTCAGCACCGCATC-3′
(SEQ ID NO: 16)


 


HAC1-12:


5′-CAAAATTGCAAGCAAGTTAACCG-3′
(SEQ ID NO: 17)






One of the two types of cDNA fragments obtained was consistent with the genome sequence; however, the other fragment was a shortened sequence lacking part thereof, i.e., a cDNA fragment spliced by Ire1p activated by UPR. In order to obtain cDNA of the activated HAC1, a PCR cycle of 94° C. for 30 seconds, 55° C. for 1 minute, and 72° C. for 30 seconds was repeated 20 times using cDNA pool, which is deduced to contain the speHAC1F DNA primer (SEQ ID NO: 20), the bglHAC1R primer (SEQ ID NO: 21), and cDNA of the activated HAC1.










speHAC1F:









(SEQ ID NO: 20)











5′-gactagtATGACTTCCTTTTCAGCACCG-3′




 



bglHAC1R:








(SEQ ID NO: 21)











5′-cagatctTCATGACAAGAAATCATCGAAT-3′







The obtained fragment of approximately 1 kb comprised a sequence from the initiation codon to the termination codon of the activated HAC1 gene (SEQ ID NO: 22), which is equivalent to the amino acid sequence of activated Hac1p comprising 320 amino acid residues (SEQ ID NO: 23). This sequence was treated with SpeI and BglII, isolated, and then introduced into the SpeI-BglII-treated pOMexPGHy. The resulting vector was designated as pOMexPGHy/Hac1 (FIG. 3). This vector comprises an activated HAC1 gene expression unit.
EXAMPLE 4
Construction of Human RRBP1 Gene Expression Vector
The human RRBP1 gene (KIAA1398, GenBank Accession No. AB037819) provided by the Kazusa DNA Research Institute was used. In order to introduce restriction enzyme sites at both terminuses of the gene, the following DNA primers, p180 MSp-F and p180 UBg-R (SEQ ID NOs: 24 and 25), and the human RRBP1 gene were used to amplify the gene of interest by PCR via 20 cycles of 94° C. for 30 seconds, 55° C. for 1 minute, and 72° C. for 5 minutes.








p180 MSp-F:









(SEQ ID NO: 24)









5′-ATACAATACAAAGTCGAGACTAGTATGGATATTTACGACACTCAAAC



 


CTT-3′


 


p180 UBg-R:








(SEQ ID NO: 25)









5′-TCTATCCACACGGATCAGATCTTCAGACAGAGGTGCCCTCCTTTGAG



 


CTG-3′






The resulting fragment of approximately 4.5 kb was introduced into the SpeI-BamHI-digested pOMexGP1A using the BD In-Fusion Dry-Down PCR Cloning Kit (BD Science). A sequence of approximately 500 bp comprising a region encoding the initiation codon of the human RRBP1 gene was determined from the SpeI site, and a fragment of approximately 500 bp comprising a region encoding the termination codon of the human RRBP1 gene was determined from the BamHI site. The resulting vector was designated as pOMexGP1A/p180PCR. pOMexGP1A/p180PCR was digested with NdeI and AscI, and a fragment comprising a region from 110 bp to 4541 bp in ORF of the human RRBP1 gene was removed from the plasmid. Separately, the human RRBP1 gene provided by the Kazusa DNA Research Institute was digested with NdeI and AscI, a fragment comprising a region from 110 bp to 4541 bp in ORF was isolated, and the isolated fragment was introduced into the aforementioned NdeI-AscI-digested pOMexGP1A/p180PCR. The resulting vector was designated as pOMexGP1A/p180 (FIG. 3). pOMexGP1A/p180 is the human RRBP1 gene expression vector.
EXAMPLE 5
Preparation of Antibody-Expressing Yeast Strain (O. minuta)
Using the NotI-digested pOMexGAT-G/Ab vector, the O. minuta YK-3 strains (Δoch1Δpep4Δprb1Δyps1Δura3Δade1: described in WO 2003/091431) were transformed via electroporation. The conditions for electroporation described in WO 2003/091431 were employed. The transformed cells were selected in YPD agar plate medium containing 50 μg/ml of G418 and cultured. Thereafter, the genomes were extracted, introduction of a heavy chain was confirmed via PCR using the aforementioned Xb-KREAEA-Hc DNA primer (SEQ ID NO: 12) and Hc-R-Bg primer (SEQ ID NO: 13), and introduction of a light chain was confirmed via PCR using the aforementioned Xb-KREAEA-Lc-F primer (SEQ ID NO: 14) and Lc-R-Ap primer (SEQ ID NO: 15). The strain in which introduction of the heavy chain and the light chain genes had been observed was designated as the antibody-producing O. minuta AO1 strain.
EXAMPLE 6
Preparation of Antibody-Producing Yeast Strain (O. minuta) that Expresses the Activated HAC1 Gene and the RRBP1 Gene
Sse8783I-digested pOMexGP1A/p180 was introduced into the antibody-producing O. minuta AO1 strain grown in Example 5 via electroporation. Transformed strain was obtained by selecting an ADE+ strain in SD agar plate medium, culturing the strain, extracting the genome, and confirming introduction of the RRBP1 gene via PCR using p180 MSp-F (SEQ ID NO: 24) and p180 UBg-R (SEQ ID NO: 25) mentioned above. The obtained transformed strain was designated as the O. minuta AK2R strain. At the same time, transformation was carried out using Sse8783I-digested pOMexGP1A to obtain the O. minuta AK2A strain as a control. Further, Aor51HI-digested pOMexPGHy-Hac1 was introduced into the O. minuta AK2R strain and the O. minuta AK2A strain via electroporation. Introduction of the activated HAC1 gene into the transformed strain was confirmed by selecting a strain in a YPD agar plate medium comprising hygromycin at 50 μg/ml, culturing the strain, extracting the genome, and confirming via PCR using the speHAC1F DNA primer (SEQ ID NO: 20) and the bglHAC1R primer (SEQ ID NO: 21). The resulting strains were designated as the O. minuta AK3RH strain and the O. minuta AK3AH strain. At the same time, Aor51 HI-digested pOMexPGHy was introduced into the O. minuta AK2R strain and the O. minuta AK2A strain to obtain the O. minuta AK3RHy strain and the O. minuta AK3AHy strain as controls.
EXAMPLE 7
Confirmation of Secretion of Antibody by Transformed Yeast Strain (O. minuta)
O. minuta AK3RH, O. minuta AK3AH, O. minuta AK3RHy, and O. minuta AK3AHy strains were cultured in BYPMG medium (1% yeast extract (Difco), 2% polypeptone (Difco), 1.5% methanol, 0.5% glycerol, and 0.1M phosphate buffer (pH 6.0)) at 28° C. for 4 days. A culture supernatant was prepared from the culture solution and subjected to SDS-PAGE. The separated protein was blotted on a PVDF membrane, and Western analysis was performed using labeled anti-human antibodies (anti-human Fc antibody and anti-human κ antibody). As a result, the strain into which the RRBP1 gene and the activated HAC1 gene had been introduced was found to secrete a significantly greater amount of antibodies than other strains, as shown in FIG. 4.
EXAMPLE 8
Productivity of Secretory Antibody by Transformed Yeast Strain (O. minuta)
The culture solution prepared in Example 7 was subjected to HPLC using Protein A columns (Poros A 50 um 4.6 mm D/50 mml, Applied Biosystems) to measure the amount of antibody production (separation conditions: equilibration buffer: 10 mM phosphate buffer (pH 6.0); elution buffer: 10 mM phosphate buffer (pH 3.4); flow rate: 4 ml/min; detection: 210 nm). As a standard sample, an antibody produced in animal cells (CHO) was used. FIG. 5 shows the antibody productivity per OD 600=1. The amounts of antibodies secreted in the O. minuta AK3RHy strain into which only the RRBP1 gene had been introduced and in the O. minuta AK3AH strain into which only the activated HAC1 gene had been introduced tended to increase, compared with the amount in the control strain, i.e., the O. minuta AK3AHy strain. The amount of antibody production in the O. minuta AK3RH strain in which both the activated HAC1 gene and the RRBP1 gene were expressed, however, was significantly greater than that in the control strain, i.e., the O. minuta AK3AHy strain, and in the strain into which either the activated HAC1 gene or the RRBP1 gene had been solely introduced. It was thus confirmed that coexpression of the activated HAC1 gene and the RRBP1 gene would produce effects greater than the synergistic effects on antibody productivity.
EXAMPLE 9
Antibody Production Under Conditions in which O-Sugar Chain Formation Using the Transformed Yeast Strain (O. minuta) is Inhibited
O. minuta AK3RH and O. minuta AK3AHy were cultured in BYPG medium (1% yeast extract (Difco), 2% polypeptone (Difco), 0.5% glycerol, and 0.1M phosphate buffer (pH 6.0)) for 2 days, and these strain were cultured in BYPM media [1% yeast extract (Difco), 2% polypeptone (Difco), 1.5% methanol, and 0.1M phosphate buffer (pH 6.0)] to which 0 μM, 1 μM, 5 μM, 10 μM, 20 μM, and 50 μM PMT inhibitors (the rhodanine-3-acetic acid derivative: 5-[[3,4-(1-phenylmethoxy)phenyl]methylene]-4-oxo-2-thioxo-3-thiazolidineacetic acid (compound (1c) described in Bioorganic & Medicinal Chemistry Letters, Vol. 14, p. 3975, 2004)) had been added at 28° C. for 2 days. A culture supernatant was prepared from the culture solution and subjected to SDS-PAGE. Thereafter, the separated protein was blotted on a PVDF membrane, and Western analysis was carried out using a labeled anti-human antibody (anti-human Fc antibody). FIG. 6 shows the results thereof. The adequate concentration of the PMT inhibitor to be added was 5 μM. In such a case, the amount of antibody secretion and the percentage of aggregate formation were increased.
EXAMPLE 10
Construction of Antibody Gene Expression Vector
In order to express a fusion protein of the secretion signal of S. cerevisiae-derived MF alpha1 (GenBank accession number: P01149) (hereafter referred to as a “aMF secretion signal”), the light chain of the anti-TRAIL receptor antibody, and the heavy chain thereof, the aMF secretion signal gene was ligated to the anti-TRAIL receptor antibody gene (WO 2001/083560) via overlap extension PCR using the following oligonucleotide primers.
For aMF Secretion Signal—a Heavy Chain of Anti-Trail Receptor Antibody










EcoALF:









(SEQ ID NO: 26)











5′-GGAATTCATGAGATTTCCTTCAAT-3′




 



AlfH02:








(SEQ ID NO: 27)











5′-CTCCACCAGCTGTACTTCTCTTTTCTCGAGAGATA-3′




 



AlfH03:








(SEQ ID NO: 28)











5′-TATCTCTCGAGAAAAGAGAAGTACAGCTGGTGGAG-3′




 



AlfH04:








(SEQ ID NO: 29)











5′-GGTCGACTCATTTACCCGGGGACAG-3′







For aMF Secretion Signal—a Light Chain of Anti-TRAIL Receptor Antibody











EcoALF:









(SEQ ID NO: 26)











5′-GGAATTCATGAGATTTCCTTCAAT-3′




 



AlfL02:








(SEQ ID NO: 30)











5′-TGGGTCATCTGAATGTCTCTTTTCTCGAGAGATA-3′




 



AlfL03:








(SEQ ID NO: 31)











5′-TATCTCTCGAGAAAAGAGACATTCAGATGACCCA-3′




 



AlfL04:








(SEQ ID NO: 32)











5′-GGTCGACCTAACACTCTCCCCTGT-3′







The aMF secretion signal gene region was amplified using, as a template, the genomic DNA of S. cerevisiae prepared using the Y-DER yeast DNA extraction reagent (PIERCE). The aMF secretion signal for the heavy chain was obtained via PCR using the EcoALF primer (SEQ ID NO: 26) and the AlfH02 primer (SEQ ID NO: 27) at 95° C. for 10 seconds, 55° C. for 30 seconds, and 68° C. for 60 seconds, and this cycle was repeated 30 times. The aMF secretion signal for the light chain was obtained via PCR using the EcoALF primer (SEQ ID NO: 26) and the AlfL02 primer (SEQ ID NO: 30) at 95° C. for 10 seconds, 55° C. for 30 seconds, and 68° C. for 60 seconds, and this cycle was repeated 30 times. The amplified target DNA fragments of approximately 0.26 kb were recovered.
The antibody gene region was amplified using cDNA of the anti-TRAIL receptor antibody (WO2001/083560) as a template. The heavy chain fragment was obtained via PCR using the AlfH03 primer (SEQ ID NO: 28) and the AlfH04 primer (SEQ ID NO: 29) at 95° C. for 10 seconds, 55° C. for 30 seconds, and 68° C. for 90 seconds, and this cycle was repeated 30 times. The light chain fragment was obtained via PCR using the AlfL03 primer (SEQ ID NO: 31) and the AlfL04 primer (SEQ ID NO: 32) at 95° C. for 10 seconds, 55° C. for 30 seconds, and 68° C. for 90 seconds, and this cycle was repeated 30 times. The target DNA fragment of the amplified heavy chain region of approximately 1.35 kb and that of the light chain region of approximately 0.65 kb were recovered.
Subsequently, aMF secretion signal region for the heavy chain and the heavy chain region of approximately 1.35 kb were used as templates to carry out PCR using the EcoALF primer (SEQ ID NO: 26) and the AlfH04 primer (SEQ ID NO: 29) at 95° C. for 10 seconds, 55° C. for 30 seconds, and 68° C. for 90 seconds, and this cycle was repeated 30 times. The amplified target DNA fragment of approximately 1.6 kb was recovered. Also, aMF secretion signal region for the light chain and the light chain region of approximately 0.65 kb were used as templates to carry out PCR using the EcoALF primer (SEQ ID NO: 26) and the AlfL04 primer (SEQ ID NO: 32) at 95° C. for 10 seconds, 55° C. for 30 seconds, and 68° C. for 60 seconds, and this cycle was repeated 30 times. The amplified target DNA fragment of approximately 0.9 kb was recovered. The recovered DNA fragments were cloned into pCR2.1-TOPO. Based on the nucleotide sequences of the insertion DNA fragments, these sequences were found to have the genes in which the aMF secretion signal had been fused in-frame to the antibody heavy chain and the aMF secretion signal had been fused in-frame to the antibody light chain, respectively. The obtained plasmids were designated as TOPO-alfHc and TOPO-alfLc, respectively. The EcoRI restriction enzyme site introduced into the EcoALF primer (SEQ ID NO: 26) and the SalI restriction enzyme sites introduced into the AlfH04 primer (SEQ ID NO: 29) and the AlfL04 primer (SEQ ID NO: 32) were used to recover DNA fragments encoding the fusion product of aMF secretion signal and the antibody heavy chain and that of the aMF secretion signal and the antibody light chain via EcoRI-SalI digestion from TOPO-alfHc and TOPO-alfLc.
In order to express the antibody heavy chain and the antibody light chain in S. cerevisiae, DNA fragments encoding the fusion product of aMF secretion signal and the antibody heavy chain and the fusion product of aMF secretion signal and the antibody light chain recovered via EcoRI-SalI digestion were ligated to the EcoRI-SalI site in the glyceraldehyde-3-phosphate dehydrogenase gene (TDH3, GAP) promoter-terminator cassette, which had been introduced into the YEp352 E. coli-yeast shuttle vector (Yeast 2, p. 163-167, 1986). The resulting plasmids were designated as YEp352GAP-II-alfHc and YEp352GAP-II-alfLc, respectively. Subsequently, the BamHI restriction enzyme sites at the both terminuses of the GAP promoter-terminator cassette were used to recover a gene fragment encoding BamHI-the GAP promoter-the aMF secretion signal-the antibody heavy chain-the GAP terminator-BamHI (fragment 1) and a gene fragment encoding BamHI-the GAP promoter-the aMF secretion signal-the antibody light chain-the GAP terminator-the BamHI (fragment 2) from YEp352GAP-II-alfHc and YEp352GAP-II-alfLc, respectively. Fragments 1 and 2 were introduced into the BamHI site of YEp352GAP-II-alfHc or Ep352GAP-II-alfLc via 3 fragment ligation, from which fragment 1 or 2 had been cleaved. The resulting vector was designated as YEp352 GAP-II-alfHc/alfLc (FIG. 7). Based on restriction enzyme cleavage patterns, tandem introduction of fragments 1 and 2 in the forward direction into YEp352 GAP-II-alfHc/alfLc was confirmed. YEp352 GAP-II-alfHc/alfLc is an antibody expression vector carrying both antibody heavy chain and light chain expression units.
EXAMPLE 11
Construction of S. cerevisiae Activated HAC1 Gene Expression Vector
RNase activity of activated IRE1 removes 252 nucleotides from HAC1 precursor mRNA of S. cerevisiae to form mature HAC1 mRNA. This mature HAC1 mRNA is translated into activated HAC1 from which 10 amino acid residues are removed from the C terminus and to which 18 amino acid residues are newly added (PNAS 97, pp. 4660-4665, 2000). Thus, a gene encoding activated HAC1 was constructed via overlap extension PCR using the following oligonucleotide primers.








HAC-Sac-ATG:









(SEQ ID NO: 33)









5′-GGAGCTCATGGAAATGACTGATTTTG-3′



 


HAC-internalR:








(SEQ ID NO: 34)









5′-GAATTCAAACCTGACTGCGCTTCTGGATTACGCCAATTGTCAAG-3′



 


HAC-internalF:








(SEQ ID NO: 35)









5′-CTTGACAATTGGCGTAATCCAGAAGCGCAGTCAGGTTTGAATTC-3′



 


HAC-Sma-STOP:








(SEQ ID NO: 36)









5′-GCCCGGGTCATGAAGTGATGAAGAAATC-3′







Genomic DNA of S. cerevisiae prepared with the use of the Y-DER yeast DNA extraction reagent (PIERCE) was used as a template. PCR was carried out using the HAC-Sac-ATG primer (SEQ ID NO: 33) and the HAC-internalR primer (SEQ ID NO: 34) at 95° C. for 10 seconds, 55° C. for 30 seconds, and 68° C. for 60 seconds, and this cycle was repeated 30 times. Separately, PCR was carried out using the HAC-internalF primer (SEQ ID NO: 35) and the HAC-Sma-STOP primer (SEQ ID NO: 36) at 95° C. for 10 seconds, 55° C. for 30 seconds, and 68° C. for 60 seconds, and this cycle was repeated 30 times. The amplified target DNA fragments of approximately 0.66 kb (fragment A) and of approximately 0.06 kb (fragment B) were recovered.
Subsequently, the amplified fragment A and fragment B were used as templates to carry out PCR using the HAC-Sac-ATG primer (SEQ ID NO: 33) and the HAC-Sma-STOP primer (SEQ ID NO: 36) at 95° C. for 10 seconds, 55° C. for 30 seconds, and 68° C. for 60 seconds, and this cycle was repeated 30 times to obtain the amplified target DNA fragment of approximately 0.7 kb. The recovered DNA fragment was cloned into pCR2.1-TOPO. Based on the nucleotide sequence of the insertion DNA fragment, the fragment was found to have a gene encoding activated HAC1 comprising 238 amino acid residues. The plasmid was designated as TOPO-aHac1. The SacI restriction enzyme site that had been introduced into the HAC-Sac-ATG primer (SEQ ID NO: 33) and the SmaI restriction enzyme site that had been introduced into the HAC-Sma-STOP primer (SEQ ID NO: 36) were used to recover a gene encoding activated HAC1 via SacI-SmaI digestion.
In order to express activated HAC1 in S. cerevisiae, a gene encoding activated HAC1 recovered via SacI-SmaI digestion was ligated to the SacI-SmaI site in the glyceraldehyde-3-phosphate dehydrogenase gene (TDH3, GAP) promoter-terminator cassette, which had been introduced into the YEp351 E. coli-yeast shuttle vector (Yeast 2, pp. 163-167, 1986). The resulting plasmid was designated as YEp351GAP-II-aHAC1 (FIG. 7). This vector comprises an activated HAC1 gene expression unit.
EXAMPLE 12
Construction of Human RRBP1 Gene Expression Vector
The human RRBP1 gene (KIAA1398, GenBank Accession No: AB037819) provided by the Kazusa DNA Research Institute was used. In order to introduce restriction enzyme sites at the both terminuses of the above gene, the gene was amplified via PCR using the following oligonucleotide primers, P180kpnatg and P180xbastop (SEQ ID NOs: 37 and 38) and the human RRBP1 gene at 95° C. for 10 seconds, 55° C. for 30 seconds, and 68° C. for 6 minutes, and this cycle was repeated 30 times.









P180kpnatg:




5′-GGGTACCATGGATATTTACGACACTC-3′
(SEQ ID NO: 37)


 


P180xbastop:


5′-GTCTAGATCAGACAGAGGTGCCCTCC-3′
(SEQ ID NO: 38)






The resulting fragment of approximately 4.7 kb was recovered and cloned into pCR2.1-TOPO. Based on the nucleotide sequence of the insertion DNA fragment, regions of approximately 600 bp at the both terminuses of the insertion fragment were confirmed to properly comprise the target nucleotide sequences. The resulting plasmid was designated as TOPO-P180. Subsequently, TOPO-P180 was digested with NdeI and HpaI restriction enzymes, and a fragment containing a region equivalent to a region between 110 bp and 4524 bp of KIAA1398 was removed. Into the removed region, a NdeI-HpaI fragment of approximately 4.4 kb of KIAA1398 was introduced to construct TOPO-P180N, and the XbaI restriction enzyme site was demethylated with the use of the E. coli SCS110 strain (Stratagene). The KpnI-XbaI restriction enzyme sites that had been introduced into the P180 kpnatg primer (SEQ ID NO: 37) and the P180xbastop primer (SEQ ID NO: 38) were used to recover a KpnI-XbaI fragment comprising a gene encoding RRBP1.
In order to express RRBP1 in S. cerevisiae, the KpnI-XbaI fragment recovered above was ligated to the KpnI-XbaI site in the glyceraldehyde-3-phosphate dehydrogenase gene (TDH3, GAP) promoter-terminator cassette, which had been introduced into the YEp352 E. coli-yeast shuttle vector (Yeast 2, pp. 163-167, 1986). The resulting plasmid was designated as YEp352GAP-II-p180. Subsequently, YEp352GAP-II-p180 was digested with the PvuI restriction enzyme, and a PvuI fragment containing the GAP promoter-the RRBP1 gene-GAP terminator was recovered. The PvuI fragment was ligated to a PvuI fragment comprising a marker gene, i.e., an essential region for replication of the YEp351 E. coli-yeast shuttle vector (Yeast 2, pp. 163-167, 1986) to construct YEp351GAP-II-p180 (FIG. 7). This vector comprises the RRBP1 gene expression unit.
EXAMPLE 13
Construction of Coexpression Vector for Activated HAC1 Gene and RRBP1 Gene
In order to introduce the activated HAC1 gene and the RRBP1 gene with the use of a single vector into S. cerevisiae, a coexpression vector for the activated HAC1 gene and the RRBP1 gene was constructed. YEp351GAP-II-aHAC1 constructed in Example 11 was digested with the HpaI restriction enzyme. Subsequently, a BamHI fragment containing the GAP promoter-the RRBP1 gene-GAP terminator was recovered from YEp352GAP-II-p180 constructed in Example 12, the both terminuses were blunt-ended using T4 DNA polymerase (Takara Bio), and the resultant was introduced into the HpaI site of YEp351GAP-II-aHAC1. The resulting plasmid was designated as YEp351GAP-II-aHAC1/p180 (FIG. 7). The nucleotide sequence was analyzed to determine the direction for introducing the BamHI fragment containing the introduced GAP promoter-the RRBP1 gene-GAP terminator. This vector comprises an expression unit for the activated HAC1 gene and the RRBP1 gene.
EXAMPLE 14
Construction of Antibody Expressing Yeast Strain and Antibody Expressing Yeast Strain (S. cerevisiae) that Expresses the Activated HAC1 Gene and the RRBP1 Gene
Competent cells of the S. cerevisiae BY4741 strains (MATaΔhis3Δleu2Δmet15Δura3) were prepared using the Frozen-EZ yeast transformation II kit (ZYMO RESARCH). The S. cerevisiae BY4741 strains were inoculated into 5 ml of YPAD medium (YPD medium containing 0.04% of adenine (Sigma)), and yeast cells obtained via overnight culture (30° C. at 310 rpm) were used. The expression vectors constructed in Example 10 to Example 13 were introduced into the S. cerevisiae BY4741 strains using the Frozen-EZ yeast transformation II kit (ZYMO RESARCH). Transformants grown on ST agar medium comprising 2% agar (the yeast nitrogen base and ammonium sulfate medium comprising 2% glucose, 0.04% adenine, and 0.3 M KCl and lacking uracil and leucine (Sigma)) were selected as antibody expressing yeast strains.
YEp352 GAP-II-alfHc/alfLc comprising antibody heavy chain and light chain expression units has the URA3 marker gene that complements uracil-requiring mutation of a host. YEp351GAP-II (a control vector into which no gene has been introduced), YEp351GAP-II-aHAC1 (an activated HAC1 expression vector), YEp351GAP-II-p180 (an RRBP1 expression vector), and YEp351GAP-II-aHAC1/p180 (a coexpression vector for the activated HAC1 gene and RRBP1) each comprise a LEU2 marker gene that complements leucine-requiring mutation of a host. Thus, these vectors were transformed into a host, so that genes could be grown only when both vectors were introduced in combination as shown below to construct four types of antibody expressing yeast strains.
S. cerevisiae T2K01 YEp352 GAP-II-alfHc/alfLc (URA3)YEp351GAP-II (LEU2)
S. cerevisiae T2K02 YEp352 GAP-II-alfHc/alfLc (URA3)YEp351GAP-II-aHAC1 (LEU2)
S. cerevisiae T2K03 YEp352 GAP-II-alfHc/alfLc (URA3)YEp351GAP-II-p180 (LEU2)
S. cerevisiae T2K04 YEp352 GAP-II-alfHc/alfLc (URA3)YEp351GAP-II-aHAC1/p180 (LEU2)
EXAMPLE 15
Antibody Productivity by Transformed Yeast Strain (S. cerevisiae)
The S. cerevisiae T2K01, S. cerevisiae T2K02, S. cerevisiae T2K03, and S. cerevisiae T2K04 strains prepared in Example 14 were cultured using ST medium at 30° C. for 3 days. The culture solution was inoculated into YPAD medium to result in a final concentration of 5% therein, and culture was conducted at 30° C. for 3 days. A culture supernatant was prepared from the culture solution, and the resultant was designated as a sample containing antibodies secreted and produced by yeast. The secreted and produced antibodies were subjected to quantitative assay via sandwich ELISA. TRAIL receptor proteins that were antigens of the anti-TRAIL receptor antibodies were adsorbed on a 96-well plate, a yeast sample was added, and detection was carried out using a peroxidase-labeled human IgG specific Fc antibody (Peroxidase-labeled affinity purified antibody to human IgG (Fc) (KPL)) and the ABTS peroxidase substrate (KPL). Antibodies produced in animal cells (NS0) were used as standard samples.
As shown in FIG. 8A, the S. cerevisiae T2K03 strain into which only the RRBP1 gene had been introduced was not substantially different from a control strain, i.e., the S. cerevisiae T2K01 strain, in terms of productivity. In the case of the S. cerevisiae T2K02 strain into which only the activated HAC1 gene had been introduced, however, productivity was approximately twice that of the control strain, i.e., the S. cerevisiae T2K01 strain. Further, antibody productivity in the S. cerevisiae T2K04 strain in which the activated HAC1 gene and the RRBP1 gene were coexpressed was significantly higher than that in the control strain, i.e., the S. cerevisiae T2K01 strain, and a strain into which the activated HAC1 gene or RRBP1 gene had been solely introduced (i.e., approximately seven times higher than the control). This indicates that coexpression of the activated HAC1 gene and the RRBP1 gene would produce effects equivalent to or greater than synergistic effects on antibody productivity.
EXAMPLE 16
Antibody Productivity Under Conditions in which Formation of O-Sugar Chain Using Transformed Yeast Strain (S. cerevisiae) is Inhibited
The S. cerevisiae T2K01, S. cerevisiae T2K02, S. cerevisiae T2K03, and S. cerevisiae T2K04 strains prepared in Example 14 were cultured using ST medium at 30° C. for 3 days. The culture solution was inoculated at a final concentration of 5% in YPAD medium to which 10 μM of PMT inhibitor (the rhodanine-3-acetic acid derivative: 5-[[3,4-(1-phenylmethoxy)phenyl]methylene]-4-oxo-2-thioxo-3-thiazolidineacetic acid (compound (1c) described in Bioorganic & Medicinal Chemistry Letters, Vol. 14, p. 3975, 2004) had been initially added, and culture was conducted at 30° C. for 3 days. A culture supernatant was prepared from the culture solution, and the resultant was designated as a sample containing antibodies secreted and produced by yeast. In the same manner as in Example 15, the sample was subjected to quantitative assay via sandwich ELISA using antibodies produced in animal cells (NS0) as standard samples.
As shown in FIG. 8B, antibody productivity of the S. cerevisiae T2K02 strain into which the activated HAC1 gene had been introduced and that of the S. cerevisiae T2K03 strain into which the RRBP1 gene had been introduced were apparently higher than that in the control strain, i.e., the S. cerevisiae T2K01 strain. Further, the amount of antibodies produced by the S. cerevisiae T2K04 strain in which the activated HAC1 gene and the RRBP1 gene were coexpressed was significantly higher than that in the control strain, i.e., the S. cerevisiae T2K01 strain, and a strain into which the activated HAC1 gene or RRBP1 gene had been solely introduced (i.e., approximately eight times higher than the control). The synergistic effects of coexpression of the activated HAC1 gene and the RRBP1 gene on antibody production were further enhanced by inhibition of O-sugar chain formation.
EXAMPLE 17
Preparation of Ogataea minuta Protease YPS1 Gene-Deficient Strain (Δoch1Δyps1Δura3Δade1)
The YPS1 gene-deficient vector, pDOMYP1, disclosed in WO 2003/091431 was cleaved with BamHI and ClaI and transformed into the O. minuta TK5-3 strain (Δoch1Δura3Δade1) disclosed in WO 2003/091431 via an electric pulse method. In order to confirm that the YPS1 genes of such genes were disrupted, the following primers were synthesized.











DY5;





5′-CTCAAGGGCCTGGAGACTACG-3′
(SEQ ID NO: 49)



 



DY3;



5′-CGGGATTCCCGAGTCGCTCACC-3′
(SEQ ID NO: 50)






Chromosome DNA isolated from the transformed strain was used as a template to carry out PCR using the DY5 and DY3 primers at 94° C. for 30 seconds, 60° C. for 1 minute, and 72° C. for 2 minutes, and this cycle was repeated 25 times. An amplified 3.7-kb DNA fragment was detected from a strain into which the plasmid had been introduced in its YPS1 locus. The selected strain was designated as the O. minuta YK4 strain (Δoch1Δura3Δade1Δyps1::URA3). After the O. minuta YK4 strain was cultured in YPD medium to a stationary phase, strains exhibiting resistance to 5-fluoroorotic acid (5-FOA) were obtained. Chromosome DNA of the 5-FOA-resistant strain was used as a template to carry out PCR using the DY5 and DY3 primers at 94° C. for 30 seconds, 60° C. for 1 minute, and 72° C. for 3 minutes, and this cycle was repeated 25 times. An amplified 1.2-kb DNA fragment was detected from an URA3-lacking strain. This Δoch1Δura3Δade1Δyps1 strain was designated as O. minuta YK5 strain.
EXAMPLE 18
Preparation of Synthetic Antibody Gene Producing Strain (O. minuta) and Antibody Production
(1) Construction of Synthetic Antibody Gene Expression Vector
pOMexGP1U disclosed in WO 2003/091431 was cleaved with SpeI, blunt-ended, and then ligated. The SalI site and the EcoT22I site of the resulting plasmid were subjected to linker change with the SpeI site and the BamHI site, respectively. The resulting plasmid was designated as pOMexGP1UΔSp.
A gene was designed from the amino acid sequence of the anti-TRAIL receptor antibody gene (WO2002/094880) while taking the frequency of the use of codons of O. minuta into consideration, and an antibody gene was artificially synthesized therefrom (Takara Bio). The S. cerevisiae SUC2 signal or chicken lysozyme signal was added to the N-terminus of the light chain and heavy chain genes, and nucleotide sequences of the restriction enzyme sites (the XbaI site on the 5′ side and the BamHI site on the 3′ site) were added to the both terminuses (nucleotide sequences: SEQ ID NOs: 51, 53, 55, and 57; amino acid sequences: SEQ ID NOs: 52, 54, 56, and 58). Two types of light chain gene fragments having different signals that had been digested with XbaI and BamHI were introduced into the pOMexGP1A vector prepared in Example 1 (2). The resulting vectors were designated as pOMexGPA/AbSUC and pOMexGPA/AbLys. Two types of heavy chain gene fragments emitting different signals that had been digested with XbaI and BamH were introduced into the SpeI-BamHI site of the pOMexGP1UΔSp vector. The resulting vectors were designated as pOMexGPUΔSp/AbSUC and pOMexGPUΔSp/AbLys.
pOMexPGHy (Example 1 (3-4)) was used as a template to carry out PCR using the PGKHy-F DNA primer (SEQ ID NO: 59) and the PGKHy-R DNA primer (SEQ ID NO: 60) at 94° C. for 30 seconds, 55° C. for 30 seconds, and 72° C. for 1 minute, and this cycle was repeated 20 times. Thus, the hygromycin B-resistant gene was amplified.









PGKHy-F:







(SEQ ID NO: 59)









5′-ATAGAACTAGCAACTAGATGAAAAAGCCTGAACTCAC-3′


 



PGKHy-R:







(SEQ ID NO: 60)









5′-CAAATCCCACGGATCACTATTCCTTTGCCCTCGGAC-3′






The amplified gene fragment was introduced into SpeI-BglII-digested pOMexPGHy using the in-fusion kit (BD Bioscience), and the nucleotide sequence of the insertion fragment was determined. The resulting plasmid was used as a template to carry out PCR using the PGKpUC-p DNA primer (SEQ ID NO: 61) and the PGKpUC-t DNA primer (SEQ ID NO: 62) at 94° C. for 30 seconds, 55° C. for 30 seconds, and 72° C. for 2 minutes, and this cycle was repeated 20 times. Thus, a gene fragment containing the PGK promoter-hygromycin B-resistant gene-PGK terminator was amplified.









PGKpUC-p:







(SEQ ID NO: 61)









5′-AATTCGAGCTCGGTACAGGGATACATGGGATACCAAAG-3′


 



PGKpUC-t:







(SEQ ID NO: 62)









5′-GAGGATCCCCGGGTACCAGGGTCGATTTTCTTGGTCGA-3′






The amplified gene fragment was introduced into Asp718I-digested pUC118 (Takara Bio) using the in-fusion kit (BD Bioscience), and the nucleotide sequence of the insertion fragment was determined. The resulting plasmid was designated as PGKHyg/pUC118. pOMexGP1UΔSp was digested with HindIII and KpnI, a cassette comprising the GAP promoter-terminator was isolated, and the isolated cassette was inserted into the HindIII-KpnI-digested PGKHyg/pUC118. The resulting plasmid was designated as GAP/HyG/pUC118. Subsequently, pUC19 (Takara Bio) was digested with NdeI and EcoRI, blunt-ended, and then ligated to remove an NdeI-EcoRI region inside pUC19. This plasmid was digested with HindIII-SacI, and a gene fragment comprising the GAP promoter-terminator isolated from GAP/HyG/pUC118 via HindIII-SacI digestion and the PGK promoter-hygromycin B-resistant gene-PGK terminator was introduced. The resulting plasmid was designated as pOMexHy.
The antibody heavy chain gene fragment to which the XbaI-BamH-digested chicken lysozyme signal had been added was introduced into the SpeI-BamHI-treated pOMexHy, and the resulting vector was designated as pOMexHy/AbLys.
(2) Preparation of Antibody Gene Expressing Yeast Strain
The NotI-digested antibody expression vectors, i.e., pOMexGPA/AbLys and pOMexGPUΔSp/AbLys, were used to transform the O. minuta YK5 strain (Δoch1Δyps1Δura3Δade1) via electroporation. The conditions for electroporation described in WO 2003/091431 were employed. The transformed cells were selected in SD agar plate medium (2% glucose, 0.67% yeast nitrogen base (Difco)). A single colony was cultured in B2YP4G medium (1.34% yeast nitrogen base (Difco), 2% yeast extract (Difco), 4% polypeptone (Difco), 4% glycerol, and 0.1M phosphate buffer (pH 6.0)) at 27° C. for 4 days. A culture supernatant was prepared from the culture solution, Western analysis was carried out in the manner as described in Example 7 to select an antibody producing strain into which the antibody light chain and heavy chain genes had been introduced, and the resulting strain was designated as the O. minuta AA1 strain. As shown in FIG. 9, the amount of antibodies secreted by the O. minuta AA1 strain was significantly greater than that secreted by the O. minuta AO1 strain prepared in Example 5.
The NotI-digested antibody expression vectors, i.e., pOMexGPA/AbSUC and pOMexGPUΔSp/AbSUC, were introduced into the O. minuta TK5-3 strain (Δoch1Δura3Δade1) described in WO 2003/091431 to obtain an antibody expressing O. minuta YY1 strain. Methods of electroporation and strain selection described above were employed.
EXAMPLE 19
Acquisition of Activated HAC1 Gene of P. pastoris and Construction of Expression Vector
The activated HAC1 gene of P. pastoris was obtained from the cells (GS115 strain of P. pastoris) in the same manner as in Example 3. cDNA was synthesized from the GS115 strain of P. pastoris in the same manner as in Example 3. This cDNA was amplified via PCR using the HACp1-1 DNA primer (SEQ ID NO: 63) and the HACp1-12 primer (SEQ ID NO: 64) shown below at 94° C. for 30 seconds, 52° C. for 30 seconds, and 72° C. for 1 minute, and this cycle was repeated 30 times. The amplified product was cloned into pCR2.1-TOPO (Invitrogen), and nucleotide sequences derived from the two types of PCR-amplified gene fragments were confirmed (SEQ ID NOs: 65 and 66).







(SEQ ID NO: 63)










HACp1-1:
5′-ATGCCCGTAGATTCTTCTCATAAGACAGC-3′


 







(SEQ ID NO: 64)










HACp1-12:
5′-CAAAGTCATTTAAATCAAATGCATTAGCGG-3′






One (SEQ ID NO: 65) of the nucleotide sequences of the obtained two types of cDNA fragment was consistent with the genomic sequence; however, the other sequence (SEQ ID NO: 66) was partially deficient and shortened. This indicates that such deficient sequence was a cDNA fragment that had been spliced by UPR-activated Ire1p. In order to obtain full-length cDNA of activated HAC1, PCR was carried out at 94° C. for 30 seconds, 55° C. for 30 seconds, and 72° C. for 1 minute using the speHACp1F DNA primer (SEQ ID NO: 67) and the bglHACp1R DNA primer (SEQ ID NO: 68) shown below and a cDNA pool that was considered to contain the activated cDNA of HAC1 described above, and this cycle was repeated 20 times.







(SEQ ID NO: 67)








speHACp1F:
5′-gactagtATGCCCGTAGATTCTTCTCATA-3′


 







(SEQ ID NO: 68)








bglHACp1R:
5′-cagatctCTATTCCTGGAAGAATACAAAGT-3′






The resulting fragment of approximately 1 kb contained a region between the initiation codon and the termination codon of the activated HAC1 gene of P. pastoris (SEQ ID NO: 69), which is equivalent to the amino acid sequence of activated Hac1p comprising 304 amino acid residues (SEQ ID NO: 70). The resultant was treated with SpeI and BglII, isolated, and then introduced into the SpeI-BglII-treated pOMexPGHy (Example 1 (3-4)). The resulting vector was designated as pOMexPGHy/PpHac1. This vector comprises the P. pastoris-derived activated HAC1 gene expression unit.
EXAMPLE 20
Construction of S. cerevisiae Activated HAC1 Gene Expression Vector
TOPO-aHac1 containing the activated HAC1 gene of S. cerevisiae prepared in Example 11 was used as a template to carry out PCR using the ScHAC-XbaF DNA primer (SEQ ID NO: 71) and the ScHAC-BamR DNA primer (SEQ ID NO: 72) at 94° C. for 30 seconds, 55° C. for 30 seconds, and 72° C. for 1 minute, and this cycle was repeated 20 times. Thus, the activated HAC1 gene of S. cerevisiae was amplified.









ScHAC-XbaF:







(SEQ ID NO: 71)









5′-gtctagaATGGAAATGACTGATTTTGAACT-3′


 



ScHAC-BamR:







(SEQ ID NO: 72)









5′-cggatccTCATGAAGTGATGAAGAAATCAT-3′






The resultant was digested with XbaI and BamHI, and a gene encoding S. cerevisiae-derived activated HAC1 was recovered. After isolation, the gene was introduced into the SpeI-BglII-treated pOMexPGHy (Example 1 (3-4)). The resulting vector was designated as pOMexPGHy/ScHac1. This vector comprises the S. cerevisiae-derived activated HAC1 gene expression unit.
EXAMPLE 21
Production of Antibody by the O. minuta Strain into which the O. minuta, P. pastoris, and S. cerevisiae Activated Hac1 Genes have been Introduced
(1) Preparation of the O. minuta Strain into which the O. minuta, P. pastoris, and S. cerevisiae Activated HAC1 Genes have been Introduced
The Aor51HI-digested O. minuta-derived activated HAC1 gene expression vector; i.e., pOMexPGHy/Hac1, the P. pastoris-derived activated HAC1 gene expression vector; i.e., pOMexPGHy/PpHac1, and the S. cerevisiae-derived activated HAC1 gene expression vector; i.e., pOMexPGHy/ScHac1, were introduced into the antibody-producing O. minuta AA1 strains grown in Example 18 via electroporation. Introduction of the activated HAC1 gene into the transformed strain was confirmed by selecting strains in YPD agar plate medium to which hygromycin B had been added at a concentration of 50 μg/ml, culturing the same, and then extracting the genome. The strain into which the O. minuta-derived activated HAC1 gene expression vector, pOMexPGHy/Hac1, had been introduced was subjected to PCR using the speHAC1F DNA primer (SEQ ID NO: 20) and the bglHAC1R DNA primer (SEQ ID NO: 21) described in Example 3. The strain into which the P. pastoris-derived activated HAC1 gene had been introduced was subjected to PCR using the speHACp1F DNA primer (SEQ ID NO: 67) and the bglHACp1R primer (SEQ ID NO: 68) at 94° C. for 30 seconds, 55° C. for 30 seconds, and 72° C. for 1 minute, and this cycle was repeated 30 times. The strain into which the S. cerevisiae-derived activated HAC1 gene had been introduced was subjected to PCR under the same conditions, except for the use of the ScHAC-XbaF DNA primer (SEQ ID NO: 71) and the ScHAC-BamR primer (SEQ ID NO: 72). The resulting strains were designated as the O. minuta AA2omH strain, the O. minuta AA2 ppH strain, and the O. minuta AA2scH strain. Simultaneously, Aor51HI-digested pOMexPGHy was introduced into the O. minuta AA1 strain to obtain the O. minuta AA2Hy strain as a control.
(2) Confirmation of Secretion of Antibody by Antibody Producing Strain into which HAC1 Gene had been Introduced
The antibody producing strains into which the HAC1 genes had been introduced prepared in (1) above; i.e., the O. minuta AA2omH strain, the O. minuta AA2 ppH strain, the O. minuta AA2scH strain, and the O. minuta AA2Hy strain, were cultured in the manner described in Example 18 (2), and Western analysis was carried out under non-reducing conditions. The results are as shown in FIG. 10. That is, addition of a sugar chain to the antibody molecule was more significantly observed but the effects of remarkable acceleration of secretion of antibody-H2L2 aggregates were not observed in the O. minuta AA2omH strain, O. minuta AA2 ppH strain, and O. minuta AA2scH strains, compared with the control O. minuta AA2Hy strain prepared in (1) above. Thus, similar effects of production could be expected via introduction of the HAC1 gene derived from a species different from a host.
(3) Productivity of Secretory Antibody by Antibody-Producing O. minuta Strain into which the HAC1 Gene had been Introduced (Quantification via TR-FRET-Based Homogeneous Analysis)
20 mM Tris-HCl buffer (pH 7.2) comprising 12 μl of 0.97 μg/ml LANCE Eu-W1024 labeled anti-human IgG (PerkinElmer), 8.3 ug/ml of biotin-conjugated mouse anti-human IgG (BD Bioscience), 16.7 ug/ml Surelight APC streptavidin (PerkinElmer), and 10% of Block Ace (Dainippon Pharmaceutical) was applied to a 96-well half area plate (Corning), and 2 μl of a sample solution prepared by adequately diluting the culture solution prepared in (2) above with 20 mM Tris-HCl buffer (pH 7.2) containing 10% of Block Ace was introduced therein, followed by agitation. The sample solution was subjected to the reaction at room temperature in the dark for 1 hour, and fluorescence was then assayed using EnVision (PerkinElmer). The amount of aggregate antibodies produced was determined based on the value at 665 nm/615 nm. Antibodies produced in animal cells (CHO) were used as standard samples. As shown in FIG. 11, no significant acceleration of antibody secretion was observed in the O. minuta AA2omH strain, the O. minuta AA2 ppH strain, and the O. minuta AA2scH strain into which the activated HAC1 genes had been introduced, compared with the O. minuta AA2Hy strain. However, similar effects of production could be expected even via introduction of the HAC1 gene derived from a species different from a host.
(4) Production of Antibody by the O. minuta Strain into which the HAC1 Gene had been Introduced Using PMT Inhibitor (1c)
A platinum loopful of the O. minuta AA2omH strain, the O. minuta AA2 ppH strain, the O. minuta AA2scH strain, and the O. minuta AA2Hy strain was inoculated into 5 ml of B2YP4G medium, cultured at 27° C. for 1 day, and then diluted with B2YP4G medium to adjust OD600 at 10. The PMT inhibitor described in Example 9 (1c: concentration of stock solution: 10 mM) was added thereto to a concentration of 2 μM therein. Culture was conducted at 27° C. for an additional 3 days, OD600 was assayed every 24 hours, and 0.04 μM each PMT inhibitor (1c) was added as the OD600 value increased by 1. A culture supernatant was prepared from the culture solution, and the amount of antibody production was measured by the method described in Example 21 (3). The results are shown in FIG. 11. It was found that the O. minuta AA2omH strain, the O. minuta AA2 ppH strain, and the O. minuta AA2scH strain into which the activated HAC1 gene had been introduced can exhibit more significant effects of accelerating antibody secretion by culturing them with the addition of the PMT inhibitor, compared with the control O. minuta AA2Hy strain.
Thus, effects of accelerating antibody secretion were found to be attained with the use of HAC1 genes of different species.
EXAMPLE 22
Isolation of O. minuta-Derived PMT Gene
The O. minuta-derived PMT1 gene was obtained via PCR using chromosome DNA of the O. minuta IFO10746 strain as a template and the PM1-5 DNA primer (SEQ ID NO: 73) and the PM1-3 DNA primer (SEQ ID NO: 74) at 94° C. for 30 seconds, 55° C. for 1 minute, and 72° C. for 2 minutes, and this cycle was repeated 25 times.







(SEQ ID NO: 73)










PM1-5:
5′-ATGGCGGGCAAAAATCAGAAATCTAGCGCG-3′


 







(SEQ ID NO: 74)










PM1-3:
5′-TTACAACTCGTCTTTGACTAGAGGCGGGGA-3′






The amplified DNA fragment of approximately 2.4 kb was recovered and cloned using the TOPO TA Cloning Kit. Plasmid DNA was isolated from the resulting clone, and the nucleotide sequence of the insertion fragment (SEQ ID NO: 75) was determined. Thus, a clone having a nucleotide sequence encoding the amino acid sequence (SEQ ID NO: 76), which is highly homologous to the amino acid sequence of the S. cerevisiae-derived PMT1 gene was selected from the insertion DNA fragment of the plasmid. The isolated plasmid was designated as pOmPM1.
The O. minuta-derived PMT2 gene was obtained using the PM2-5 DNA primer (SEQ ID NO: 77) and the PM2-3 DNA primer (SEQ ID NO: 78), the PMT4 gene was obtained using the PM4-5 DNA primer (SEQ ID NO: 79) and the PM4-3 DNA primer (SEQ ID NO: 80), the PMT5 gene was obtained using the PM5-5 DNA primer (SEQ ID NO: 81) and the PM5-3 DNA primer (SEQ ID NO: 82), and the PMT6 gene was obtained using the PM6-5 DNA primer (SEQ ID NO: 83) and the PM6-3 DNA primer (SEQ ID NO: 84), in the same manner as in the case of the PMT1 gene. Plasmids comprising the PMT2 gene (nucleotide sequence: SEQ ID NO: 85; amino acid sequence: SEQ ID NO: 86), the PMT4 gene (nucleotide sequence: SEQ ID NO: 87; amino acid sequence: SEQ ID NO: 88), the PMT5 gene (nucleotide sequence: SEQ ID NO: 89; amino acid sequence: SEQ ID NO: 90) and the PMT6 gene (nucleotide sequence: SEQ ID NO: 91; amino acid sequence: SEQ ID NO: 92) were designated as pOmPM2, pOmPM4, pOmPM5, and pOmPM6, respectively.







(SEQ ID NO: 77)










PM2-5:
5′-ATGGGCGAACGTACGGGCAAAAGTGCGCTC-3′


 







(SEQ ID NO: 78)










PM2-3:
5′-CTAATCGGAAATTCTCCACGTGCTCAAGAG-3′


 







(SEQ ID NO: 79)










PM4-5:
5′-ATGGGGCCCAAAATAAAGACCGGCAAGAAA-3′


 







(SEQ ID NO: 80)










PM4-3:
5′-CTATTTAGCAAAATGCAGTTTGATGTTGAG-3′


 







(SEQ ID NO: 81)










PM5-5:
5′-ATGGACGAGAAAAACATCTCTGGCTTAGAA-3′


 







(SEQ ID NO: 82)










PM5-3:
5′-CTACTCACTATAGACGGAGCAGTCGATCGA-3′


 







(SEQ ID NO: 83)










PM6-5:
5′-ATGTCCGAGTCAGAGCTGAGAAACCGCAAA-3′


 







(SEQ ID NO: 84)










PM6-3:
5′-CTAAGCTATACGCCAGGTGGAAACCCAGTT-3′






EXAMPLE 23
Preparation of PMT Gene-Insertionally Inactivating or Disrupting Vector for O. minuta 
(1) Preparation of PMT Geneinsertional Inactivation Vector
(1-1) Preparation of PMT1 Gene-Insertional Inactivation Vector
In order to isolate a partial sequence of the PMT1 gene with the use of pOmPM1 obtained in Example 22, the following PMT1hIII DNA primer (SEQ ID NO: 93) and the PMT1Kp DNA primer (SEQ ID NO: 94) were used to carry out PCR at 94° C. for 30 seconds, 55° C. for 30 seconds, and 72° C. for 2 minutes, and this cycle was repeated 15 times.







(SEQ ID NO: 93)










PMT1hIII:
5′-caagcttGGACCTACAACACGTCCGAAGAA-3′


 







(SEQ ID NO: 94)










PMT1Kp:
5′-cggtaccGGTTTGATACCTTGGGTGGCACA-3′






The amplified DNA fragment of approximately 1.6 kb was digested with HindIII and KpnI and recovered. Subsequently, pPICZα (Invitrogen) was digested with BglII and blunt-ended. The HindIII linker was inserted therein, further digested with BamHI, and blunt-ended, followed by insertion of the KpnI linker. The resulting plasmid was designated as pZ-Hd-Kp. pZ-Hd-Kp was digested with HindIII and KpnI, a 2.0-kb DNA fragment containing a zeocine-resistant gene was isolated, and the PCR-amplified partial sequence of the PMT1 gene was inserted therein. The nucleotide sequence of the partial sequence of the inserted PMT1 gene was determined, and the resulting plasmid was then designated as pOmPM1dZ. pOmPM1dZ is capable of insertionally inactivating the structural gene (CDS) region and the promoter region of the O. minuta PMT1 gene and inhibiting transcription of the PMT1 gene. Separately, pOMexGPUΔSp prepared in Example 18 was digested with HindIII and KpnI, a gene fragment containing the GAP promoter and the terminator was recovered, and the recovered fragment was inserted into the HindIII-KpnI-digested 2.0-kb DNA fragment containing the zeocine-resistant gene. The resulting plasmid was designated as GAP/Z.
(1-2) Preparation of PMT2 Gene-Insertional Inactivation Vector
In order to isolate a partial sequence of the PMT2 gene with the use of pOmPM2 obtained in Example 22, the following PMT2hIII DNA primer (SEQ ID NO: 95) and the PMT2Kp DNA primer (SEQ ID NO: 96) were used to carry out PCR at 94° C. for 30 seconds, 55° C. for 30 seconds, and 72° C. for 2 minutes, and this cycle was repeated 15 times.







(SEQ ID NO: 95)








PMT2hIII:
5′-gaagcttACTACATAATTCGTGTACGTGTTC-3′


 







(SEQ ID NO: 96)








PMT2Kp:
5′-cggtaccGTCGCCGTATTGGTCAGCAATCTC-3′






The amplified DNA fragment of approximately 1.5 kb was recovered and digested with HindIII and KpnI, and the digested fragment was then recovered. The obtained DNA fragment was inserted into a 2.0-kb DNA fragment containing a zeocine-resistant gene, which had been isolated from pZ-Hd-Kp via digestion with HindIII and KpnI, and the nucleotide sequence of the partial sequence of the inserted PMT2 gene was determined. The resulting plasmid was designated as pOmPM2dZ. pOmPM2dZ is capable of insertionally inactivating the structural gene (CDS) region and the promoter region of the O. minuta PMT2 gene and inhibiting transcription of the PMT2 gene.
(1-3) Preparation of PMT4 Gene-Insertional Inactivation Vector
In order to isolate a partial sequence of the PMT4 gene with the use of pOmPM4 obtained in Example 22, the following PMT4FHdinf DNA primer (SEQ ID NO: 97) and the PMT4RKpinf DNA primer (SEQ ID NO: 98) were used to carry out PCR at 94° C. for 30 seconds, 55° C. for 30 seconds, and 72° C. for 2 minutes, and this cycle was repeated 15 times.









PMT4FHdinf:







(SEQ ID NO: 97)









5′-GTCATGAGATCCaagctGATCCCTCAATGGAGATCTACT-3′


 



PMT4RKpinf:







(SEQ ID NO: 98)









5′-GGTGTGTGGGGGATCgGGATGCAAATGGATGGCTCGAAC-3′






The obtained DNA fragment of approximately 1.5 kb was inserted into a 2.0-kb DNA fragment containing a zeocine-resistant gene, which had been isolated from pZ-Hd-Kp via digestion with HindIII and KpnI using the in-fusion kit (BD Bioscience). The nucleotide sequence of the partial sequence of the inserted PMT4 gene was determined, and the resulting plasmid was then designated as pOmPM4dZ.
(2) Preparation of PMT Gene-Disrupting Vector
(2-1) Preparation of PMT5 Gene-Disrupting Vector
pROMU1 containing a gene fragment having repeat sequences of approximately 0.8 kb upstream and downstream of the URA3 structural gene of O. minuta disclosed in WO 2003/091431 was digested with HindIII, and blunt-ended, followed by insertion of the BamHI linker. The resulting vector was digested with BamHI and BglII, and a fragment of approximately 3.3 kb containing repeat sequences and the URA3 gene of O. minuta was introduced into the BamHI-digested pBluescript KS- (Stratagene). The resulting vector was designated as rURApBKS.
Chromosome DNA of the O. minuta IFO10746 strain was used as a template, the PMT5maeF2 DNA primer (SEQ ID NO: 99) and the PMT5maeR DNA primer (SEQ ID NO: 100) were used to carry out PCR at 94° C. for 30 seconds, 55° C. for 1 minute, and 72° C. for 2 minutes, and this cycle was repeated 25 times. The amplified DNA fragment of approximately 1.5 kb was recovered and introduced into the BamHI-HindIII-digested rURApBKS using the in-fusion kit (BD Bioscience). The nucleotide sequence of the inserted gene fragment was determined. The resulting vector was designated as PMT5K/O/rURA3pre.
Chromosome DNA of the O. minuta IFO10746 strain was used as a template, the PMT5ushiroF DNA primer (SEQ ID NO: 101) and the PMT5ushiroR DNA primer (SEQ ID NO: 102) were used to carry out PCR at 94° C. for 30 seconds, 55° C. for 1 minute, and 72° C. for 2 minutes, and this cycle was repeated 25 times. The amplified DNA fragment of approximately 1.5 kb was recovered and introduced into the NotI-digested PMT5K/O/rURA3pre using the in-fusion kit (BD Bioscience). The nucleotide sequence of the inserted gene fragment was determined. The resulting vector was designated as PMT5K/O/rURA3.









PMT5maeF2:







(SEQ ID NO: 99)









5′-GACGGTATCGATAAGCTTGATGCGCGGCCTTCCGACCTT-3′


 



PMT5maeR:







(SEQ ID NO: 100)









5′-CTGGGGAAGCTCGGATCCGGCTCGAGGTCTTCGTTCAGA-3′


 



PMT5ushiroF:







(SEQ ID NO: 101)









5′-CTAGTTCTAGAGCGGCCCAGGTCGCTTTCAGGCAGCAG-3′


 



PMT5ushiroR:







(SEQ ID NO: 102)









5′-CACCGCGGTGGCGGCCAAGCTTGGGTACCGGCTCGCGTAG-3′






(2-2) Preparation of PMT6 Gene-Disrupting Vector

Chromosome DNA of the O. minuta IFO10746 strain was used as a template, the PMT6inf5′armF DNA primer (SEQ ID NO: 103) and the PMT6inf5′armR DNA primer (SEQ ID NO: 104) were used to carry out PCR at 94° C. for 30 seconds, 55° C. for 2 minutes, and 72° C. for 2 minutes, and this cycle was repeated for 25 times. The amplified DNA fragment of approximately 2.8 kb was recovered and introduced into the BamHI-digested rURApBKS using the in-fusion kit (BD Bioscience). The nucleotide sequence of the inserted gene fragment was determined. The resulting vector was designated as PMT6K/O/rURA3pre.
Chromosome DNA of the O. minuta IFO10746 strain was used as a template, the PMT6inf3′armF DNA primer (SEQ ID NO: 105) and the PMT6inf3′armR2DNA primer (SEQ ID NO: 106) were used to carry out PCR at 94° C. for 30 seconds, 55° C. for 2 minutes, and 72° C. for 2 minutes, and this cycle was repeated for 25 times. The amplified DNA fragment of approximately 2.5 kb was recovered and introduced into the NotI-SacII-digested PMT6K/O/rURA3pre using the in-fusion kit (BD Bioscience). The nucleotide sequence of the inserted gene fragment was determined. The resulting vector was designated as PMT6K/O/rURA3.









PMT6inf5′armF:







(SEQ ID NO: 103)









5′-GCAGCCCGGGGgatccACGAAACCACGTCCTACT-3′


 



PMT6inf5′armR:







(SEQ ID NO: 104)









5′-GGGGAAGCTcggatcGACTCATCTTGAAACGCA-3′


 



PMT6inf3′armF:







(SEQ ID NO: 105)









5′-AGTTCTAGAGCGGCCTTACCACCATTACATGCC-3′


 



PMT6inf3′armR2:







(SEQ ID NO: 106)









5′-AATTGGAGCTCCACCGCGGCCGCAACTTACTCGACGCTAA-3′






EXAMPLE 24
Preparation of Antibody Producing O. minuta Strain with an Insertionally Inactivated or Disrupted Pmt Gene and Evaluation Thereof
(1) Preparation of Antibody Producing O. minuta Strain with an Insertionally Inactivated PMT Gene
As for the PMT gene-insertional inactivation vectors prepared in Example 23, the PMT1 gene-insertional inactivation vector was digested with PstI, the PMT2 gene-insertional inactivation vector was digested with XhoI, and the PMT4 geneinsertional inactivation vector was digested with HindIII. These digestion products were introduced into the antibody producing O. minuta YY1 strain grown in Example 18 via electroporation. Interruption of PMT genes in the transformed strains was confirmed in the following manner. Strains were selected in YPD agar plate medium to which zeocine had been added to a concentration of 50 μg/ml therein and cultured, followed by extraction of the genomes. The PMT1 gene was subjected to PCR at 94° C. for 30 seconds, 55° C. for 30 seconds, and 72° C. for 1 minutes, and this cycle was repeated 30 times, with the use of a DNA primer pair of Zeo1 (SEQ ID NO: 107) and PMT1zeo1 (SEQ ID NO: 109) and a DNA primer pair of Zeo2 (SEQ ID NO: 108) and PMT1zeo2 (SEQ ID NO: 110). The PMT2 gene was subjected to PCR under the same conditions, with the use of a DNA primer pair of Zeo1 (SEQ ID NO: 107) and PMT2zeo1 (SEQ ID NO: 111) and a DNA primer pair of Zeo2 (SEQ ID NO: 108) and PMT2zeo2 (SEQ ID NO: 112). The PMT4 gene was subjected to PCR under the same conditions, with the use of a DNA primer pair of Zeo1 (SEQ ID NO: 107) and PMT4PCR3′armF (SEQ ID NO: 113) and a DNA primer pair of Zeo2 (SEQ ID NO: 108) and PMT4PCR5′armR3 (SEQ ID NO: 114). Thus, insertional inactivation of PMT genes upon introduction of insertional inactivation vectors was confirmed.







(SEQ ID NO: 107)








Zeo1:
5′-GAACGGCACTGGTCAACTTGGCCAT-3′


 







(SEQ ID NO: 108)








Zeo2:
5′-CTTCGTGGCCGAGGAGCAGGACTGA-3′


 







(SEQ ID NO: 109)








PMT1zeo1:
5′-GAATTCTAGCCGAGCATGAGCTA-3′


 







(SEQ ID NO: 110)








PMT1zeo2:
5′-CGTTCAGACTCTTGTTGATTTTCCAC-3′


 







(SEQ ID NO: 111)








PMT2zeo1:
5′-GCTGTGCCACTGCACGCCTCGACTC-3′


 







(SEQ ID NO: 112)








PMT2zeo2:
5′-CTTGTCCCTCTTGAATGGCGAGTG-3′


 







(SEQ ID NO: 113)








PMT4PCR3′armF:
5′-GGAACACGCCAAACATCATG-3′


 







(SEQ ID NO: 114)








PMT4PCR5′armR3:
5′-CACAAGCAGAATCAGGCAC-3′






The resulting strains were designated as the O. minuta YY2P1 strain (a strain with an insertionally inactivated PMT1 gene), the O. minuta YY2P2 strain (a strain with an insertionally inactivated PMT2 gene), and the O. minuta YY2P4 strain (a strain with an insertionally inactivated PMT4 gene). Also, Sse8387I-digested GAP/Z was introduced into the O. minuta YY1 strain, and a zeocine-resistant strain was selected to obtain the O. minuta YY2Z strain as a control strain.
(2) Productivity of Secretory Antibody by Antibody Producing O. minuta Strain with an Insertionally Inactivated PMT Gene
The antibody producing strains with an insertionally inactivated PMT genes prepared in (1) above were cultured in the manner as described in Example 18 (2), and Western analysis was then carried out. The results are shown in FIG. 12 and FIG. 14. That is, the amount of secretion of antibody aggregates was slightly increased from that attained by the O. minuta YY2P1 strain with an insertionally inactivated PMT1 gene, the O. minuta YY2P2 strain with an insertionally inactivated PMT2 gene, and the O. minuta YY2P4 strain with an insertionally inactivated PMT4 gene, compared with the O. minuta YY2Z strain prepared in (1) above (FIG. 12: lane 3 and lane 6; FIG. 14: lane 3).
(3) Productivity of Secretory Antibody by the Antibody Producing O. minuta Strain with an Insertionally Inactivated Pmt Gene (Quantification Via TR-FRET-Based Homogeneous Analysis)
The amount of antibodies produced in the culture solution prepared in (2) above was measured by the method described in Example 21 (3). As a standard sample, antibodies produced in animal cells (CHO) were used. As shown in FIG. 13 and FIG. 15, the amounts of antibody aggregates secreted were slightly increased in the O. minuta YY2P1 strain with an an insertionally inactivated PMT1 gene, the O. minuta YY2P2 strain with an insertionally inactivated PMT2 gene, and the O. minuta YY2P4 strain with an insertionally inactivated PMT4 gene, compared with the O. minuta YY2Z strain prepared in (1) above (FIG. 13: YY2P1 and YY2P2; FIG. 15: YY2P4).
(4) Preparation of PMT5- and PMT6-Deficient Strains and Evaluation Thereof
(4-1) Preparation of O. minuta PMT5 Gene-Deficient Strain (Δoch1Δyps1Δura3Δade1Δpmt5)
The PMT5 gene-disrupting vector, PMT5K/O/rURA3, prepared in Example 23 (2-1) was digested with HindIII and transformed into the Ogataea minuta YK5 strain (Δoch1Δura3Δade1Δyps1) prepared in Example 17 via the electric pulse method. In order to confirm that the PMT5 genes of these strains had been disrupted, the following primers were synthesized.







(SEQ ID NO: 115)










gPMT5-5:
5′-CGGTGACGACTTCGACTAGTCGAG-3′


 







(SEQ ID NO: 116)










gPMT5-2:
5′-CGGTGCTGTTGGCGTCGTCATGGGTG-3′


 







(SEQ ID NO: 117)










gPMT5-3:
5′-GGCGCGTTCCAATTCCACTCTGCTG-3′


 







(SEQ ID NO: 118)










gPMT5-4:
5′-CGACGAGTCCTCTCACCAGGAGGTTG-3′






Chromosome DNA isolated from the transformed strain was used as a template to carry out PCR using the gPMT5-5 primer (SEQ ID NO: 115) and the gPMT5-2 primer (SEQ ID NO: 116) at 94° C. for 30 seconds, 60° C. for 1 minute, and 72° C. for 2 minutes, and this cycle was repeated 25 times. A 4.9-kb amplified DNA fragment was detected from a strain into which a plasmid had been incorporated in its PMT5 locus. Similarly, chromosome DNA isolated from the transformed strain was used as a template to carry out PCR using the gPMT5-3 primer (SEQ ID NO: 117) and the gPMT5-4 primer (SEQ ID NO: 118) at 94° C. for 30 seconds, 60° C. for 1 minute, and 72° C. for 2 minutes, and this cycle was repeated 25 times. A 4.9-kb amplified DNA fragment was detected from a strain into which a plasmid had been incorporated in its PMT5 locus. The selected strain was designated as the O. minuta YK6 strain (Δoch1Δura3Δade1Δyps1Δpmt5::URA3).
(4-2) Preparation of O. minuta PMT6 Gene Deficient Strain (Δoch1Δyps1Δura3Δade1Δpmt6)
The PMT6 gene-disrupting vector, PMT6K/O/rURA3, prepared in Example 23 (2-2) was digested with BamHI and NotI and then transformed into the O. minuta YK5 strain (Δoch1Δura3Δade1Δyps1) prepared in Example 17 via the electric pulse method. In order to confirm that the PMT6 genes of these strains had been disrupted, the following primers were synthesized.







(SEQ ID NO: 119)










PMT6 PCR3′armF:
5′-TGTGGGTGCGATCCTGAG-3′


 







(SEQ ID NO: 120)










PMT6 PCR3′armR:
5′-GCCGTCGTTGGAGCAAAACT-3′


 







(SEQ ID NO: 121)










PMT6 PCR5′armF:
5′-GCATGTGCCACTGCTAAA-3′


 







(SEQ ID NO: 122)










PMT6 PCR5′armR:
5′-GACCAACTTTCCCGTGTAA-3′






Chromosome DNA isolated from the transformed strain was used as a template, the PMT6 PCR3′armF primer (SEQ ID NO: 119) and the PMT6 PCR3′armR primer (SEQ ID NO: 120) were used to carry out PCR at 94° C. for 30 seconds, 60° C. for 1 minute, and 72° C. for 2 minutes, and this cycle was repeated for 25 times. A 5.8-kb amplified DNA fragment was detected from a strain into which a plasmid had been incorporated in its PMT6 locus. Similarly, chromosome DNA isolated from the transformed strain was used as a template to carry out PCR using the PMT6 PCR5′armF primer (SEQ ID NO: 121) and the PMT6 PCR5′armR primer (SEQ ID NO: 122) at 94° C. for 30 seconds, 60° C. for 1 minute, and 72° C. for 2 minutes, and this cycle was repeated 25 times. A 6.3-kb amplified DNA fragment was detected from a strain into which a plasmid had been incorporated in its PMT6 locus. The selected strain was designated as the O. minuta YK7 strain (Δoch1Δura3Δade1Δyps1Δpmt6::URA3).
(5) Preparation of Antibody Producing O. minuta Strain in which the PMT5 and PMT6 Genes had been Disrupted
The NotI-digested antibody expression vector, pOMexGPA/AbLys, (prepared in Example 18 (1)) and the Sse8387I-digested antibody expression vector, pOMexHy/AbLys, (prepared in Example 18 (1)) were used to transform the O. minuta YK6 strain (Δoch1Δyps1Δura3Δade1Δpmt5::rURA3) and the O. minuta YK7 strain (Δoch1Δyps1Δura3Δade1Δpmt6::rURA3) via electroporation. The transformed cells were selected in SD agar plate medium (2% glucose, 0.67% yeast nitrogen base (Difco)) to which hygromycin B had been added at a concentration of 50 μg/ml therein. A single colony was cultured in B2YP4G medium (1.34% yeast nitrogen base (Difco), 2% yeast extract (Difco), 4% polypeptone (Difco), 4% glycerol, 0.1M phosphate buffer (pH 6.0)) at 27° C. for 4 days. A culture supernatant was prepared from the culture solution, Western analysis was carried out by the method described in Example 7, and antibody-producing strains into which the antibody light chain and heavy chain genes had been introduced were selected. The resulting strains were designated as the O. minuta AP5 strain (the PMT5 gene-deficient strain) and the O. minuta AP6 strain (the PMT6 gene-deficient strain). Separately, the NotI-digested antibody expression vector, pOMexGPA/AbLys, and the Sse8387I-digested pOMexHy/AbLys were introduced into the O. minuta YK4 strain (Δoch1Δura3Δade1Δyps1::rURA3) via electroporation, and antibody producing strains were prepared and selected as control strains in the manner described above. The obtained strains were designated as O. minuta Acon strains.
(6) Productivity of Secretory Antibody by an Antibody Producing O. minuta Strain in which the PMT5 and PMT6 Genes had been Disrupted
The O. minuta AP5 strain in which the PMT5 gene had been disrupted and the O. minuta AP6 strain and the O. minuta Acon strain in which the PMT6 gene had been disrupted were cultured in the manner described in Example 18 (2), and non-reducing Western analysis was carried out. The amount of antibodies produced in the culture solution was measured in the manner described in Example 21 (3). As a standard sample, antibodies produced in animal cells (CHO) were used. The results are shown in FIG. 16. That is, there was no significant difference in productivity of antibody aggregates between the O. minuta AP5 strain or O. minuta AP6 strain and the control O. minuta Acon strain.
EXAMPLE 25
Preparation of the O. minuta Strain with an Insertionally Inactivated PMT Gene and with the HAC1 Gene Introduced Therein and Evaluation Thereof
(1) Preparation of the O. minuta Strain with an Insertionally Inactivated PMT Gene the HAC1 Gene had been Introduced
The O. minuta-derived activated HAC1 gene expression vector, pOMexPGHy/Hac1, prepared in Example 3 was digested with Aor51HI and introduced into the O. minuta YY2P2 strain (a strain with an insertionally inactivated PMT2 gene), the O. minuta YY2P4 strain (a strain with an insertionally inactivated PMT4 gene), and the O. minuta YY2Z strain (the control strain), which had been prepared in Example 24 (1), via electroporation. In order to confirm that the activated HAC1 gene had been introduced into the transformed strain, strains were selected in YPD agar plate medium to which hygromycin B had been added at a concentration of 50 μg/ml therein, the strains were cultured, the genomes were extracted, and PCR was carried out, in accordance with the method of Example 6. The obtained strains were designated as the O. minuta YY3P2omH strain (the strain with an insertionally inactivated PMT2 gene and with the HAC1 gene introduced therein), the O. minuta YY3P4omH strain (the strain with an insertionally inactivated PMT4 gene and with the HAC1 gene introduced therein), and the O. minuta YY3ZomH strain (the control strain into which the HAC1 gene had been introduced). Simultaneously, Aor51HI-digested pOMexPGHy was introduced into the O. minuta YY2Z strain, and the O. minuta YY3ZHy strain (the control strain into which a vector had been introduced) was obtained as a control strain.
(2) Productivity of Secretory Antibody by O. minuta Strain with an Insertionally Inactivated PMT Gene and with the HAC1 Gene Introduced Therein
The antibody producing strains with an insertionally inactivated PMT gene and with the HAC1 gene introduced therein prepared in (1) above were cultured by the method described in Example 18 (2), and Western analysis was carried out. The results are shown in FIG. 12 and FIG. 14. The O. minuta YY3P2omH strain with an insertionally inactivated PMT2 gene and with the HAC1 gene introduced therein was found to secrete a significantly greater amount of antibodies than the O. minuta YY3ZomH strain into which only the HAC1 gene had been introduced and the O. minuta YY2P2 strain with an insertionally inactivated PMT2 gene (FIG. 12: lane 7). The O. minuta YY3P4omH strain with an insertionally inactivated PMT4 gene and with the HAC1 gene introduced therein was found to secrete a significantly greater amount of antibodies than the O. minuta YY3ZomH strain into which only the HAC1 gene had been introduced and the O. minuta YY2P4 strain with an insertionally inactivated PMT4 gene (FIG. 14: lane 6).
The amount of antibodies produced in the culture solution was measured by the methods described in Example 8 and in Example 21 (3). As a standard sample, antibodies produced in animal cells (CHO) were used. The results are shown in FIG. 13 and in FIG. 15. The O. minuta YY3P2omH strain with an insertionally inactivated PMT2 gene and with the HAC1 gene introduced therein was found to secrete a significantly greater amount of antibody aggregates than the control strains, i.e., the O. minuta YY3ZomH strain (a strain into which only the HAC1 gene had been introduced), the O. minuta YY2P2 strain (a strain with an insertionally inactivated PMT2 gene), and the O. minuta YY3ZHy strain (a control strain into which a vector had been introduced) (FIG. 13: O. minuta YY3P2omH strain). Also, the O. minuta YY3P4omH strain with an insertionally inactivated PMT4 gene and with the HAC1 gene introduced therein was found to secrete a significantly greater amount of antibodies than the control strains, i.e., the O. minuta YY3ZomH strain (a strain into which only the HAC1 gene had been introduced), the O. minuta YY2P4 strain (a strain with an insertionally inactivated PMT4 gene), and the O. minuta YY3ZHy strain (a control strain into which a vector had been introduced) (FIG. 15: the O. minuta YY3P4omH strain).
EXAMPLE 26
Antibody Production by O. minuta Strain with an Insertionally Inactivated PMT Gene and with the HAC1 Gene Introduced Therein Using the PMT Inhibitor (1c)
A platinum loopful of antibody-producing strains with an insertionally inactivated PMT gene and with the HAC1 gene introduced therein prepared in Example 25 (1) was inoculated into 5 ml of B2YP4G medium, cultured at 27° C. for 1 day, and diluted with B2YP4G medium to adjust OD600 to 10. The resultant was added to the PMT inhibitors described in Example 9 (1c; concentrations of stock solutions: 0.1 mM, 0.5 mM, 2.5 mM, and 10 mM) to concentrations of 0.016 μM, 0.08 μM, 0.4 μM, and 2.0 μM, respectively. Culture was conducted at 27° C. for an additional 3 days, OD600 was measured every 24 hours, and PMT inhibitors (1c) were added in amounts of 0.00032 μM, 0.0016 μM, 0.008 μM, and 0.04 μM, respectively, as the OD600 value increased by 1. A culture supernatant was prepared from the culture solution, and Western analysis was carried out by the method described in Example 18 (2). The results are shown in FIG. 17. Further, productivity of secretory antibodies was quantified by the method described in Example 21 (3). The results are shown in FIG. 18. The highest antibody productivity in the O. minuta YY3P2omH strain (a strain with an insertionally inactivated PMT2 gene and with the HAC1 gene introduced therein) was attained when approximately 0.008 μM PMT inhibitor (concentration of stock solution: 2.5 mM) was added as the OD600 value increased by 1. The highest antibody productivity in the O. minuta YY3P4omH strain (a strain with an insertionally inactivated PMT4 gene and with the HAC1 gene introduced therein) was attained when approximately 0.04 μM PMT inhibitor (concentration of stock solution: 10 mM) was added as the OD600 value increased by 1. It can be deduced that sugar chain addition is strongly inhibited by inhibition of PMT gene expression and use of PMT inhibitors in combination. In order to inhibit sugar chain addition regarding the strain into which HAC1 had been introduced, a higher productivity of aggregate antibodies can be expected by inhibition of PMT protein activity with inhibition of PMT gene expression and use of PMT inhibitors in combination.
EXAMPLE 27
Evaluation of PMT Inhibitor (5a)
A platinum loopful of the O. minuta YY1 strains into which antibody genes had been introduced was inoculated into 5 ml of B2YP4G medium, cultured at 27° C. for 1 day, and diluted with B2YP4G medium to adjust OD600 at 10. A PMT inhibitor ({(5Z)-4-oxo-5-[3-(1-phenylethoxy)-4-(2-phenylethoxy)benzylidene]-2-thioxo-1,3-thiazolidin-3-yl}acetic acid (compound 5a described in Bioorganic & Medicinal Chemistry Letters, Vol. 14, p. 3975, 2004) that is different from the PMT inhibitor (1c) described in Example 9 was added thereto to result in a concentration of 1 μm therein. Culture was conducted at 27° C. for an additional 3 days, OD600 was measured every 24 hours, and the PMT inhibitor was added in amounts of 0.02 μM as the OD600 value increased by 1. A culture supernatant was prepared from the culture solution, Western analysis was carried out by the method described in Example 18 (2), and productivity of secretory antibodies was quantified by the method described in Example 21 (3). The results are shown in FIG. 19 and in FIG. 20. Productivity of antibody aggregates was considered to tend to increase via the addition of the PMT inhibitor (5a), which had been newly examined. Thus, the PMT inhibitor (5a) was considered to have effects equivalent to those of the PMT inhibitor (1c) described in Example 9.
INDUSTRIAL APPLICABILITY
The present invention enables high-level secretory production of proteins having complicated structures, such as antibodies, as well as general proteins, in yeast.

## Claims

1. An isolated polynucleotide encoding an activated Homologous to atf/Creb 1 (HAC 1)-protein, wherein the isolated polynucleotide is selected from (a) and (b):
(a) a polynucleotide comprising a cDNA sequence that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 23; and
(b) a polynucleotide comprising a cDNA sequence that encodes a polypeptide having at least 80% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 23, wherein said polypeptide having at least 80% amino acid identity to the amino acid sequence of SEQ ID NO: 23 has function of activating the unfolded protein response (UPR).

2. A method for producing a transformed yeast cell, comprising the step of introducing the isolated polynucleotide according to claim 1 into a yeast cell.

3. The method according to claim 2, wherein the yeast cell is a methanol-assimilating yeast cell.

4. The method according to claim 3, wherein the methanol-assimilating yeast cell is Ogataea minuta.

5. The method according to claim 2, wherein the yeast cell is Saccharomyces cerevisiae.

6. An expression vector comprising the polynucleotide according to claim 1.

7. The expression vector according to claim 6, which is pOMexPGHy/Hac1.

8. The expression vector according to claim 6, which further comprises a polynucleotide encoding a mammalian ribosome-binding protein 1 (RRBP 1) protein.

9. The expression vector according to claim 8, wherein the polynucleotide encoding a mammalian RRBP 1 protein is selected from (a) and (b):
(a) a polynucleotide encoding human RRBP 1 or dog RRBP 1; and
(b) a polynucleotide encoding a polypeptide having at least 80% amino acid sequence identity to the amino acid sequence of human RRBP 1 or dog RRBP 1, wherein said polypeptide having at least 80% amino acid identity to the amino acid sequence of human RRBP 1 or dog RRBP 1 has a function of stabilizing mRNA.

10. A transformed yeast cell comprising the expression vector according to claim 6.

11. The transformed yeast cell according to claim 10, wherein the endogenous protein-O-mannosyltransferase (PMT) gene of said transformed yeast cell is further insertionally inactivated.

12. The transformed cell according to claim 11, wherein the yeast cell is Ogataea minuta.

13. A method for producing a protein, comprising the step of culturing the transformed yeast cell according to claim 10 in a medium under conditions in which O-sugar chain synthesis is inhibited, wherein said transformed yeast cell further expresses a polynucleotide encoding a foreign protein to produce the foreign protein; and isolating the produced foreign protein from the culture medium.

14. The method for producing a protein according to claim 13, wherein O-sugar chain synthesis is inhibited by insertionally inactivating the endogenous protein O-mannosyltransferase (PMT) gene of said yeast cell.

15. The method for producing a protein according to claim 13, wherein O-sugar chain synthesis is inhibited by adding an inhibitor of the endogenous PMT protein of said yeast cell to the medium.

16. The method according to claim 15, wherein the inhibitor is 5-[[3,4-(1-phenylmethoxy)phenyl]methylene]-4-oxo-2-thioxo-3-thiazolidineacetic acid or {(5Z)-4-oxo-5-3-(1-phenylethoxy)-4-(2-phenylethoxy)benzylidene]-2-thioxo-1,3-thiazolidin-3-yl}acetic acid.

17. The method for producing a protein according to claim 13, wherein O-sugar chain synthesis is inhibited by insertionally inactivating the endogenous PMT gene of said yeast cell, and further adding an inhibitor of the endogenous PMT protein of said yeast cell to the medium.

18. A transformed yeast cell comprising the expression vector according to claim 8.

19. A method for producing a protein, comprising the step of culturing the transformed yeast cell according to claim 18 in a medium under conditions in which O-sugar chain synthesis is inhibited, wherein said transformed yeast cell further expresses a polynucleotide encoding a foreign protein to produce the foreign protein; and isolating the produced foreign protein from the culture medium.

20. A transformed yeast cell comprising the expression vector of claim 9.

21. A method for producing a protein, comprising the step of culturing the transformed yeast cell according to claim 20 in a medium under conditions in which O-sugar chain synthesis is inhibited, wherein said transformed yeast cell further expresses a polynucleotide encoding a foreign protein to produce the foreign protein; and isolating the produced foreign protein from the culture medium.

22. A transformed yeast cell comprising a first isolated polynucleotide encoding an activated HAC1 protein selected from (a) and (b):
(a) a polynucleotide comprising a cDNA sequence that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 23; and
(b) a polynucleotide comprising a cDNA sequence that encodes a polypeptide having at least 80% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 23, wherein said polypeptide having at least 80% amino acid identity to the amino acid sequence of SEQ ID NO: 23 has a function of activating the unfolded protein response (UPR); and
wherein said transformed yeast cell further comprises a second isolated polynucleotide selected from (c) and (d):
(c) a polynucleotide encoding human RRBP 1 or dog RRBP 1; and
(d) a polynucleotide encoding a polypeptide having at least 80% amino acid sequence identity to the amino acid sequence of human RRBP 1 or dog RRBP1, wherein said polypeptide having at least 80% amino acid identity to the amino acid sequence of human RRBP1 or dog RRBP1 has a function of stabilizing mRNA.

23. The transformed yeast cell according to claim 22, wherein the yeast cell is a methanol-assimilating yeast cell.

24. The transformed yeast cell according to claim 23, wherein the methanol-assimilating yeast cell is Ogataea minuta.

25. The transformed yeast cell according to claim 22, wherein the yeast cell is Saccharomyces cerevisiae.

26. The transformed yeast cell according to claim 22, wherein said yeast cell further comprises a polynucleotide encoding a foreign protein.

27. The transformed yeast cell according to claim 26, wherein the foreign protein is a multimeric protein.

28. The transformed yeast cell according to claim 27, wherein the multimeric protein is a heteromultimer.

29. The transformed yeast cell according to claim 28, wherein the heteromultimer is an antibody or a functional fragment thereof.

30. A method for producing a foreign protein, comprising culturing the transformed yeast cell according to claim 26 in a medium to express the foreign protein, and isolating the produced foreign protein from the culture medium.

31. The method according to claim 30, wherein the culturing step is conducted under conditions in which the transferase activity of endogenous protein O-mannosyltransferase (PMT) of said transformed yeast cell is inhibited.

32. The method according to claim 31, wherein said transferase activity is inhibited by addition of an inhibitor of said transferase activity to the medium.