# Patent

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

## Abstract

This invention provides a means for high-level secretory production of a protein, and, in particular, a protein having a complicated structure such as an antibody, in a host cell such as a yeast cell. This invention provides a method for high-level secretory production of a foreign protein with the use of a transformed host cell having one or more types of chaperone protein genes and via suppression of O sugar chain inherent to a host cell such as a yeast cell.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a National Stage of International Application No. PCT/JP2008/070155 filed Oct. 29, 2008, claiming priority based on Japanese Patent Application Nos. 2007-283731 filed Oct. 31, 2007 and 2007-288845 filed Nov. 6, 2007, the contents of all of which are incorporated herein by reference in their entirety.
TECHNICAL FIELD
The present invention relates to a method for high-level secretory production of a protein, mainly in yeast.
BACKGROUND ART
When secretory proteins are expressed, signal sequences thereof are recognized by signal recognition particles (SRPs), and the secretory proteins then pass through the translocon and enter into the endoplasmic reticulum. When secretory proteins pass through the translocon, the higher-order structures thereof are loosened, and the proteins are folded in the endoplasmic reticulum (ER). Protein folding can spontaneously occur, and various molecular chaperones assist such folding. A native conformation formed in the endoplasmic reticulum is critical for secretion, and misfolded proteins cannot enter the secretory pathway located downstream. When proteins are not correctly folded in the endoplasmic reticulum, proteins of having abnormal higher-order structures are disadvantageously aggregated therein. Such disturbance in modification that takes place in the endoplasmic reticulum (i.e., addition of a sugar chain or a disulfide bond) and deteriorated transportation from the endoplasmic reticulum cause “endoplasmic reticulum stress.” As a means for dealing with such endoplasmic reticulum stress, a stress response referred to as “unfolded protein response (UPR)” is induced in eukaryotic cells. Transcription induction and translation regulation of UPR are responses that restore accumulated abnormal proteins. There is also a mechanism referred to as “ER-associated degradation (ERAD)” that degrades and eliminates abnormal proteins, so as to maintain homeostasis in the endoplasmic reticulum. Further, molecular chaperones that loosen the aggregated proteins for the purpose of folding are known, as well as molecular chaperones that assist protein folding in the endoplasmic reticulum. For example, HSP104 can perform a reaction that cannot be performed with the aid of other chaperones that cooperate with HSP70 and solubilizes proteins from the aggregates (Glover J R, Lindquist S, Hsp104, Hsp70, and Hsp40: A novel chaperone system that rescues previously aggregated proteins, Cell, 1998, 94:73-82).
Meanwhile, antibody molecules form aggregates of a proper conformation (H2L2) through the formation of a disulfide bond between the antibody heavy chain and the antibody light chain or between antibody heavy chains and forming an intramolecular disulfide bond of the antibody heavy chain and the antibody light chain. In the case of eukaryotic cells such as yeast cells, introduction of a disulfide bond via protein oxidative folding is carried out by the oxidative protein disulfide isomerase (PDI) in the endoplasmic reticulum (Benjamin P. Tu and Jonathan S. Weissman, Oxidative protein folding in eukaryotes: Mechanisms and consequences, J. Cell Biol., 2004, 164: 341-346). PDI that is reduced via oxidization of substrate proteins is reoxidized by oxidative ERO1 localized in the vicinity of the membrane (Mezghrani, A., Fassio, A., Benham, A., Simmen, T., Braakman, I., and Sitia, R., Manipulation of oxidative protein folding and PDI redox state in mammalian cells, EMBO. J., 2001, 20: 6288-6296 and Frand, A. R. and C. A. Kaiser, Ero1p oxidizes protein disulfide isomerase in a pathway for disulfide bond formation in the endoplasmic reticulum, Mol. Cell, 1999, 4: 469-477). In the yeast endoplasmic reticulum, there are 5 types of PDI families (i.e., PDI1, EUG1, MPD1, MPD2, and EPS1) (Per Norgaard, Vibeke Westphal, Christine Tachibana, Lene Alsoe, Bjorn Holst, Jakob R. Winther, Functional Differences in Yeast Protein Disulfide Isomerases, J. Cell Biology, 2001, 152 (3): 553-562). Among such PDI families, those that are confirmed to form an intramolecular disulfide bond with ERO1 are limited to PDI1 and MPD2. It is also reported that efficiency for protein oxidative folding is improved by BiP/Kar2, which functions in cooperation with PDI (Marcus Mayer, Ursula Kies, Robert Kammermeier, and Johannes Buchner, BiP and PDI Cooperate in the Oxidative Folding of Antibodies in Vitro, J. Biol. Chem., 2000, 275 (38): 29421-29425). BiP/Kar2 is also associated with induction by active HAC1 of various genes associated with the aforementioned UPR. Active HAC1 is activated by splicing of HAC1 by the IRE1 transmembrane kinase/nuclease. IRE1 to which BiP/Kar2 has bound is dissociated when BiP/Kar2 acts on a protein having an abnormal structure in the endoplasmic reticulum, it exhibits nuclease activity through the formation of a dimmer, and it produces active HAC1 by splicing HAC1 (Cox J S., Shamu C E., Walter P., Transcriptional induction of genes encoding endoplasmic reticulum resident proteins requires a transmembrane protein kinase, Cell, 1993, 73: 1197-1206 and Sidrauski C. and Walter P., The transmembrane kinase Ire1p is a site-specific endonuclease that initiates mRNA splicing in the unfolded protein response, Cell, 1997, 90 (6): 1031-1039). Also, Bip/Kar2 is associated with protein folding in the endoplasmic reticulum in cooperation with SCJ1 located in the endoplasmic reticulum (Susana Silberstein, Gabriel Schlenstedt, Pam A. Silver, and Reid Gilmore, A Role for the DnaJ Homologue Scj1p in Protein Folding in the Yeast Endoplasmic Reticulum, J. Cell Biol., 1998, 143 (4): 921-933).
Thus, it has been demonstrated that various molecular chaperones are associated with correct folding of secretory proteins. Also, attempts to increase the amount of secretory proteins produced with the aid of molecular chaperones have been made. When an S—S bond produces abundant human serum albumin in the yeast K. lactis, for example, the amount of production is increased by 15 times upon introduction of ERO1 or PDI1 (Tiziana Lodi, Barbara Neglia, and Claudia Donnini, Secretion of human serum albumin by Kluyveromyces lactis overexpressing K1PDI1 and K1ERO1, Applied. Environ. Microbiol., 2005, 71 (8): 4359-4363). However, simultaneous introduction of ERO1 and PDI1 does not lead to any further improvement, and it merely advances the timing of production. Also, IL-1β having no S—S bond has no effects. It has been also reported that the amount of secretory production was increased by 8 times via coexpression of RatPDI and Bip, when producing single-chain antibody fragments (scFvs) in S. cerevisiae (Shusta, E. V., Raines, R. T., Pluckthun, A., and Wittrup, K. D., Increasing the secretory capacity of Saccharomyces cerevisiae for production of single-chain antibody fragments, Nat. Biotechnol., 1998, 16: 773-777). In this report, it was concluded that Bip prevents aggregation and the amount of secretory production of PDI was increased by isomerase activity instead of chaperone activity. When producing single-chain antibody fragments (scFvs) in Pichia pastries, also, the amount of secretory production was increased by 3 times via coexpression with Bip; however, use of PDI or PDI in combination with Bip could not yield any effects (Damasceno, Leonardo, et al., Cooverexpression of chaperones for enhanced secretion of a single-chain antibody fragment in Pichia pastris. Applied. Microbio. Biotech. (2007) 74 (2): 381-389). Further, the effects of an increased PDI level on secretion of IL-15 and the tumor necrosis factor receptor (i.e., Fc fusion protein (TNFR:Fc)) were inspected in CHO cells. As a result, PDI overexpression allowed retention of TNFR:Fc (i.e., proteins rich in disulfide bonds) in the cells, but IL-15 was not retained in the cells (Davis R., Schooley K., Rasmussen B., Thomas J., Reddy P., Effect of PDI overexpression on recombinant protein secretion in CHO cells. Biotechnol. Prog., 2000, 16: 736-743). This suggests that PDI overexpression disadvantageously results in a decrease in the amount of secretory production in CHO cells.
As described above, attempts to improve secretory production of proteins such as antibodies via coexpression of molecular chaperones that assist protein folding have been made. However, effects cannot be always attained because of types or combinations of host cells or molecular chaperones. In an example of antibody production, single-stranded antibodies are merely produced, and a method for effectively producing high-molecular-weight proteins or aggregate proteins, including complete antibodies, has not yet been discovered.
DISCLOSURE OF THE INVENTION
Accordingly, the present invention is intended to provide a means for high-level secretory production of proteins and, more particularly, proteins having complicated structures, such as antibodies, in yeast or other host cells.
The present inventors have conducted concentrated studies in order to attain the above object. As a result, they discovered that coexpression of one or more types of molecular chaperone genes with the targets of expression (i.e., foreign protein genes) in yeast or other host cells would result in an increase in the amount of secretory production of foreign proteins. 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 by approximately 2 to 45 times. The present invention has been completed based on such findings.
Specifically, the present invention includes the following inventions.
(1) A transformed host cell into which one or a combination of two or more of the chaperone genes (a) to (c) below have been introduced:
(a) a gene comprising DNA which consists of the nucleotide sequence as shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, or 13;
(b) a gene which hybridizes under stringent conditions to a DNA consisting of a nucleotide sequence complementary to DNA consisting of the nucleotide sequence as shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, or 13 and encodes a protein having activity of accelerating foreign protein secretion; and
(c) a gene which consists of a nucleotide sequence having at least 80% homology with the nucleotide sequence as shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, or 13 and encodes a protein having activity of accelerating foreign protein secretion.
(2) A transformed host cell into which one or a combination of two or more of the genes encoding the chaperone proteins (d) to (f) below have been introduced:
(d) a protein which consist of the amino acid sequence as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, or 14;
(e) a protein which consist of an amino acid sequence having at least 80% homology with the amino acid sequence as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, or 14 and has activity of accelerating foreign protein secretion; and
(f) a protein which consist of an amino acid sequence derived from the amino acid sequence as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, or 14 by deletion, substitution, and/or addition of one or several amino acids and has activity of accelerating foreign protein secretion.
(3) A transformed host cell into which one or a combination of two or more of the chaperone genes (g) or (h) below have been introduced;
(g) a gene encoding PDI1, MPD1, SCJ1, ERO1, FKB2, JEM1, LHS1, MPD2, ERJ5, or EUG1 derived from S. cerevisiae or a homologous gene thereof; or
(h) a gene encoding PDI, ERO1-Lα, ERO1-Lβ, or GRP78 derived from a human, a homologous gene thereof, or a codon-modified gene thereof.
(4) A transformed host cell into which a gene selected from the group consisting of one or a combination of two or more of the chaperone genes (i) to (xii) below, a homologous gene thereof, or a codon-modified gene thereof has been introduced:
(i) a gene encoding PDI1 derived from O. minuta; 
(ii) a gene encoding PDI1 derived from S. cerevisiae; 
(iii) a gene encoding PDI derived from a human;
(iv) a gene encoding ERO1 derived from O. minuta; 
(v) a gene encoding ERO1 derived from a human;
(vi) a gene encoding Kar2 derived from O. minuta; 
(vii) a gene encoding PDI1 and a gene encoding ERO1 derived from O. minuta; 
(viii) a gene encoding PDI1 and a gene encoding Kar2 derived from O. minuta; 
(ix) a gene encoding PDI derived from a human and a gene encoding ERO1 derived from O. minuta; 
(x) a gene encoding PDI1, a gene encoding ERO1, and a gene encoding Kar2 derived from O. minuta; 
(xi) a gene encoding PDI, a gene encoding ERO1-Lβ, and a gene encoding GRP78 derived from a human; and
(xii) a gene encoding PDI derived from a human, a gene encoding ERO1 derived from O. minuta, and a gene encoding GRP78 derived from a human.
(5) The transformed host cell according to any of (1) to (4), wherein the host cell is a eukaryotic cell.
(6) The transformed host cell according to (5), wherein the eukaryotic cell is a yeast cell.
(7) The transformed host cell according to (6), wherein the yeast is methanol-assimilating yeast.
(8) The transformed host cell according to (7), wherein the methanol-assimilating yeast is Ogataea minuta. 
(9) The transformed host cell according to (6), wherein the yeast is Saccharomyces cerevisiae. 
(10) The transformed host cell according to any of (1) or (9), into which a gene encoding a foreign protein has been introduced.
(11) The transformed host cell according to (10), wherein the foreign protein is a multimeric protein.
(12) The transformed host cell according to (11), wherein the multimeric protein is a heteromultimer.
(13) The transformed host cell according to (12), wherein the heteromultimer is an antibody or a functional fragment thereof.
(14) The transformed host cell according to (10), wherein the foreign protein is glycosyltransferase.
(15) A method for producing a protein comprising culturing the transformed host cell according to any of (10) to (14) in a medium and sampling a target protein from the culture product.
(16) The method for producing a protein according to (15), wherein culture is carried out under conditions in which protein O-mannosyltransferase (PMT) activity is inhibited.
(17) The method for producing a protein according to (16), wherein protein O-mannosyltransferase (PMT) activity is inhibited with the addition of an inhibitor of PMT activity to the medium.
(18) The method for producing a protein according to (17), wherein the inhibitor of PMT activity is 5-[[3,4-(1-phenylmethoxy)phenyl]methylene]-4-oxo-2-thioxo-3-thiazolidineacetic acid.
(19) The method for producing a protein according to (16), wherein protein O-mannosyltransferase (PMT) activity is inhibited via destruction of the PMT gene.
(20) The method for producing a protein according to (16), wherein protein O-mannosyltransferase (PMT) activity is inhibited via destruction of the PMT gene and with the addition of an inhibitor of PMT activity to the medium.
(21) The method for producing a protein according to (20), wherein the inhibitor of PMT activity is 5-[[3,4-(1-phenylmethoxy)phenyl]methylene]-4-oxo-2-thioxo-3-thiazolidineacetic acid.
(22) The method for producing a protein according to any of (19) to (21), wherein destruction of the PMT gene is single destruction of the PMT5 gene or the PMT6 gene or double destruction of the PMT5 gene and the PMT6 gene.
(23) Any of the chaperone genes (a) to (c) below derived from Ogataea minuta: 
(a) a gene comprising DNA which consists of the nucleotide sequence as shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, or 13;
(b) a gene which hybridizes under stringent conditions to a DNA consisting of a nucleotide sequence complementary to DNA consisting of the nucleotide sequence as shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, or 13 and encodes a protein having activity of accelerating foreign protein secretion; and
(c) a gene which consists of a nucleotide sequence having at least 80% homology with the nucleotide sequence as shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, or 13 and encodes a protein having activity of accelerating foreign protein secretion.
(24) A gene encoding any of the chaperone proteins (d) to (f) below:
(d) a protein which consist of the amino acid sequence as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, or 14;
(e) a protein which consist of an amino acid sequence having at least 80% homology with the amino acid sequence as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, or 14 and has activity of accelerating foreign protein secretion; and
(f) a protein which consist of an amino acid sequence derived from the amino acid sequence as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, or 14 by deletion, substitution, and/or addition of one or several amino acids and has activity of accelerating foreign protein secretion.
(25) An expression vector comprising the gene according to (23) or (24).
(26) An expression vector comprising the gene (g) or (h) below:
(g) a gene encoding PDI1, MPD1, SCJ1, ERO1, FKB2, JEM1, LHS1, MPD2, ERJ5, or EUG1 derived from S. cerevisiae or a homologous gene thereof; or
(h) a gene encoding PDI, ERO1-Lα, ERO1-Lβ, or GRP78 derived from a human, a homologous gene thereof, or a codon-modified gene thereof.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the construction of expression vectors for various chaperone proteins derived from O. minuta (onaP03606, onaP09407, onaP09507, onaP09607, and onaP09707).
FIG. 2 shows the construction of the coexpression vector (onaP11107) for a human-derived chaperone protein (codon-modified Human PDI) and for an O. minuta-derived chaperone protein (OmERO1).
FIG. 3 shows the construction of the OmKar2 expression vector (pOU1/Kar2-Ppt) via regulation of the PGK promoter.
FIG. 4 shows the construction of the coexpression vector (onaP11007) for three types of chaperone proteins (OmPDI1, OmERO1, and OmKar2) derived from O. minuta. 
FIG. 5 is a chart showing the results of measuring the amount of secreted antibodies of an antibody-producing yeast strain (i.e., the ona02306 strain) cultured with or without the addition of an inhibitor of O-mannosylation (rhodanine-3-acetic acid derivative 1c) (1: 1c was not added; 2: 100 μl of medium containing 20 μM 1c was added on day 3; 3: 100 μl of medium containing 20 μM 1c was added on day 2 and day 3).
FIG. 6 shows the results of Western blot analysis of antibodies secreted in the culture supernatant of an antibody-producing yeast strain (i.e., the ona02306 strain) cultured with or without the addition of an inhibitor of O-mannosylation (rhodanine-3-acetic acid derivative 1c) (1: 1c was not added; 2: 100 μl of medium containing 20 μM 1c was added on day 3; 3: 100 μl of medium containing 20 μM 1c was added on day 2 and day 3).
FIG. 7 is a chart showing the results of measuring the amount of secreted antibodies of an antibody-producing yeast strains (cultured with or without the addition of an inhibitor of O-mannosylation (rhodanine-3-acetic acid derivative 1c)) into which gene of a single chaperone protein (SCJ1, EUG1, ERO1, Kar2, MPD1, PDI1, and HSP104) derived from O. minuta strain had been introduced.
FIG. 8 shows the results of Western blot analysis of antibodies secreted in the culture supernatant of an antibody-producing yeast strains (cultured with or without the addition of an inhibitor of O-mannosylation (rhodanine-3-acetic acid derivative 1c)) into which gene of a single chaperone protein (SCJ1, EUG1, ERO1, Kar2, MPD1, PDI1, and HSP104) derived from O. minuta strain had been introduced (top: reducing SDS-PAGE/WB; antibody Hc and antibody Lc are simultaneously detected; bottom: non-reducing SDS-PAGE/WB; antibody Lc is detected).
FIG. 9 is a chart showing the results of measuring the amount of secreted antibodies of an antibody-producing yeast strains (cultured with or without the addition of an inhibitor of O-mannosylation (rhodanine-3-acetic acid derivative 1c)) into which genes of a plurality of chaperone proteins (2×PDI1, PDI1/HSP104, PDI1/Kar2, and PDI1/ERO1) derived from O. minuta strain had been introduced.
FIG. 10 shows the results of Western blot analysis of antibodies secreted in the culture supernatant of an antibody-producing yeast strains (cultured with or without the addition of an inhibitor of O-mannosylation (rhodanine-3-acetic acid derivative 1c)) into which genes of a plurality of chaperone proteins (2×PDI1, PDI1/HSP104, PDI1/Kar2, and PDI1/ERO1) derived from O. minuta strain had been introduced (top: reducing SDS-PAGE/WB; antibody Hc and antibody Lc are simultaneously detected; bottom: non-reducing SDS-PAGE/WB; antibody Lc is detected).
FIG. 11 is a chart showing the results of measuring the amount of secreted antibodies of an antibody-producing yeast strains (cultured with or without the addition of an inhibitor of O-mannosylation (rhodanine-3-acetic acid derivative 1c)) into which genes of chaperone proteins (PDI1) derived from S. cerevisiae, a human, and a synthesized human had been introduced.
FIG. 12 is a chart showing the results of measuring the amount of secreted antibodies of an antibody-producing yeast strains (cultured with or without the addition of an inhibitor of O-mannosylation (rhodanine-3-acetic acid derivative 1c)) into which combinations of genes of three types of chaperone proteins derived from O. minuta strain (PDI1/EPO1/Kar2) and genes of chaperone proteins derived from different species (human PDI/OmERO1) had been introduced.
FIG. 13 shows construction of an antibody expression vector, YEp352 GAP-II-ScKarHc/ScKarLc.
FIG. 14 is a chart showing the results of measuring the amount of secreted antibodies of an antibody-producing yeast strains (cultured with or without the addition of an inhibitor of O-mannosylation (rhodanine-3-acetic acid derivative 1c)) into which the gene of the chaperone protein (PDI1) derived from S. cerevisiae had been introduced.
FIG. 15 shows changes in activity values for sialic acid transferase (ST3Gal1) expressed in a strain (YT-2) into which the O. minuta-derived chaperone protein (PDI1) gene was not introduced and in a strain (YT-3) into which the PDI1 gene had been introduced with the elapse of time.
FIG. 16 shows the results of Western blot analysis of sialic acid transferase (ST3Gal1) expressed in a strain (YT-2) into which the O. minuta-derived chaperone protein (PDI1) gene was not introduced and in a strain (YT-3) into which the PDI1 gene had been introduced.
FIG. 17 shows changes in activity values for sialic acid transferase (ST3Gal1) expressed in strains (YT-4, YT-5, YT-6, and YT-7) into which genes of the O. minuta-derived chaperone proteins (PDI1x2, PDI1/OmKar2, PDI1/ERO1, and PDI1/HSP104) had been introduced with the elapse of time.
FIG. 18 shows the results of Western blot analysis of sialic acid transferase (ST3Gal1) expressed in strains (YT-4, YT-5, YT-6, and YT-7) into which genes of the O. minuta-derived chaperone proteins (PDI1x2, PDI1/OmKar2, PDI1/ERO1, and PDI1/HSP104) had been introduced.
FIG. 19 is a chart showing the results of measuring the amount of secreted antibodies of an antibody-producing animal cell lines (i.e., the COS-1 cell) into which genes of the CHO-cell-derived chaperone proteins (CHOBiP and CHOPDI) and the human-derived chaperone protein (hERO1-Lβ) had been introduced.
FIG. 20 is a chart showing the results of measuring the amount of secreted antibodies of an antibody-producing yeast strains (cultured with or without the addition of an inhibitor of O-mannosylation (rhodanine-3-acetic acid derivative 1c)) into which genes of the human-derived chaperone proteins (PDI, ERO1-Lα, ERO1-Lβ, and GRP78) had been introduced.
FIG. 21 shows the results of Western blot analysis of antibodies secreted in the culture supernatant of antibody-producing yeast strains (cultured with or without the addition of an inhibitor of O-mannosylation (rhodanine-3-acetic acid derivative 1c)) into which genes of the human-derived chaperone proteins (PDI, ERO1-Lα, ERO1-Lβ, and GRP78) had been introduced.
FIG. 22 shows the results of Western blot analysis attained when yeast aMF signal-fused human lysozyme is expressed in strains into which the coexpression vector for three types of the O. minuta-derived chaperone proteins (OmPDI1, OmERO1, and OmKar2) or the coexpression vector for three types of the human-derived chaperone proteins (synthesized hPDI, synthesized hERO1-Lβ, and synthesized hGRP78) had been introduced.
FIG. 23 is a chart showing the results of measuring the amount of secreted antibodies when cultured with or without the addition of an inhibitor of O-mannosylation (rhodanine-3-acetic acid derivative 1c) in strains prepared by introducing coexpression vectors for three types of the O. minuta-derived chaperone proteins (OmPDI1, OmERO1, and OmKar2) and an antibody gene into a strain in which the PMT genes had been destroyed.
FIG. 24 is a photograph showing the results of scanning electron microscopic observation of the O. minuta strain in which the PMT gene had been destroyed.
FIG. 25 is a photograph showing the results of transmission electron microscopic observation of the O. minuta strain in which the PMT gene had been destroyed.
FIG. 26 shows the results of examining concentration of an inhibitor of O-mannosylation (rhodanine-3-acetic acid derivative 1c) that has been added to a strain prepared by introducing coexpression vectors for three types of the O. minuta-derived chaperone proteins (OmPDI1, OmERO1, and OmKar2) and an antibody gene into the O. minuta strain in which the PMT4 gene had been destroyed.
FIG. 27 shows elution patterns of size exclusion chromatography (SEC)-HPLC of various purified antibodies produced from O. minuta. 
FIG. 28 shows the results of Western blot analysis after non-reducing electrophoresis of various purified antibodies produced from O. minuta. 
FIG. 29 is a chart showing the results of measuring cytotoxic activity of various purified antibodies produced from O. minuta. 
FIG. 30 is a chart showing the results of measuring cytotoxic activity of various purified antibodies produced from O. minuta. 



This patent application claims priority from Japanese Patent Application Nos. 2007-283731 filed on Oct. 31, 2007 and 2007-288845 filed on Nov. 6, 2007, and includes part or all of the contents as disclosed in the descriptions thereof.
Hereafter, the present invention is described in detail.
1. Gene Used for High-Level Secretory Production of a Protein
High-level secretory production of a protein in the present invention involves the use of a gene encoding a set of molecular chaperones (hereafter such gene is referred to as a “chaperone gene”) that functions in protein folding and inhibition of degradation or aggregation of a denatured protein in the endoplasmic reticulum.
Examples of chaperone genes that are used in the present invention include PDI1, MPD1, SCJ1, EUG1, ERO1, HSP104, and Kar2 genes derived from the Ogataea minuta (O. minuta) strain, which was newly obtained in the present invention. The PDI1, MPD1, SCJ1, EUG1, ERO1, HSP104, and Kar2 genes derived from Ogataea minuta (O. minuta) each consists of a nucleotide sequence as shown in SEQ ID NOs: 1, 3, 5, 7, 9, 11, or 13, and amino acid sequences deduced based on such nucleotide sequences are as shown in SEQ ID NOs: 2, 4, 6, 8, 10, 12, or 14.
The chaperone gene used in the present invention may be a gene encoding a protein consisting of an amino acid sequence derived from the amino acid sequence as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, or 14 by deletion, substitution, and/or addition of one or several amino acids, provided that such gene has activity of accelerating foreign protein secretion.
The number of amino acids that may be deleted, substituted, and/or added is preferably 1 to several. The number represented by the term “several” is not particular limited. For example, such number is 20 or smaller, preferably 10 or smaller, more preferably 7 or smaller, and further preferably 5 or smaller, approximately. The term “mutation” used herein primarily refers to a mutation that is artificially introduced via a known method for preparing a mutant protein, and the term may refers to a mutation that is similar to one existing in nature.
Also, the chaperone gene used in the present invention may be a gene encoding a protein which consists of an amino acid sequence having at least 80% homology with the amino acid sequence as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, or 14 and has activity of accelerating foreign protein secretion. The term “at least 80% homology” preferably refers to at least 85% homology, more preferably at least 90% homology, 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 term “activity of accelerating foreign protein secretion” used herein refers to activity of high-level secretion of a correctly folded foreign protein in a host cell based on activity of molecular chaperones for protein folding (e.g., formation of a disulfide bond), activity of refolding a denatured protein into a normal protein, and activity of inhibiting denatured protein aggregation in the endoplasmic reticulum.
Also, the term “ . . . has activity of accelerating foreign protein secretion” refers that such activity is substantially equivalent to activity of a protein having the amino acid sequence as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, or 14.
The chaperone gene used in the present invention may be a gene which hybridizes under stringent conditions to a DNA consisting of a nucleotide sequence complementary to DNA comprising the nucleotide sequence as shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, or 13 and encodes a protein having activity of accelerating foreign protein secretion.
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 a highly homologous nucleic acid, i.e., a nucleic acid consisting of a nucleotide sequence having at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% homology to the nucleotide sequence as shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, or 13 undergo hybridization, but complementary strands of a nucleic acid having lower homology levels would not undergo hybridization. More specifically, the sodium concentration is 15 to 750 mM, preferably 50 to 750 mM, and more preferably 300 to 750 mM, the temperature is 25° C. to 70° C., preferably 50° C. to 70° C., and more preferably 55° C. to 65° C., and formamide concentration is 0% to 50%, preferably 20% to 50%, and more preferably 35% to 45%. Under stringent conditions, further, a filter is generally washed at sodium salt concentration of 15 to 600 mM, preferably 50 to 600 mM, and more preferably 300 to 600 mM, and temperature is 50° C. to 70° C., preferably 55° C. to 70° C., and more preferably 60° C. to 65° C., after hybridization.
A person skilled in the art can easily obtain such homologous genes by referring to, for example, Molecular Cloning (Sambrook, J. et al., Molecular Cloning: A Laboratory Manual 2nd ed., Cold Spring Harbor Laboratory Press, 10 Skyline Drive Plainview, N.Y., 1989). Also, nucleotide sequence homology search can be carried out via FASTA, BLAST, or other programs.
The amino acid 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 chaperone gene used in the present invention may be a chaperone gene derived from another organism species, such as other types of yeast, mold, or a human. As chaperone gene derived from another organism species, a chaperone gene derived from a human that corresponds to the aforementioned chaperone gene (the PDI1, MPD1, SCJ1, EUG1, ERO1, HSP104, or Kar2 gene) derived from Ogataea minuta is preferable. A chaperone gene other than the aforementioned chaperone genes may also be used.
Examples of chaperone genes derived from a human include PDI (GenBank Accession No. BC010859; SEQ ID NO: 140), ERO1-Lα (GenBank Accession No. AF081886; SEQ ID NO: 143), ERO1-Lβ (GenBank Accession No. BC044573; SEQ ID NO: 146), and GRP78 (GenBank Accession No. AL354710; SEQ ID NO: 149).
Also, examples of chaperone genes derived from Saccharomyces cerevisiae include PDI1 (Primary SGDID: S000000548; SEQ ID NO: 120), MPD1 (Primary SGDID: S000005814; SEQ ID NO: 122), SCJ1 (Primary SGDID: S000004827; SEQ ID NO: 124), ERO1 (Primary SGDID: S000004599; SEQ ID NO: 126), FKB2 (Primary SGDID: S000002927; SEQ ID NO: 128), JEM1 (Primary SGDID: S000003609; SEQ ID NO: 130), LHS1 (Primary SGDID: S000001556; SEQ ID NO: 132), MPD2 (Primary SGDID: S000005448; SEQ ID NO: 134), ERJ5 (Primary SGDID: S000001937; SEQ ID NO: 136), and EUG1 (Primary SGDID: S000002926; SEQ ID NO: 138). Sequence information regarding the genes derived from Saccharomyces cerevisiae is available from SGD (Saccharomyces genome database: http://www.yeastgenome.org/).
Such chaperone genes derived from other organism species may also be genes homologous thereto, provided that such genes have activity of accelerating foreign protein secretion. Extent of such sequence homology, the number of deletion, substitution, or addition of amino acids, stringent conditions, and methods for mutagenesis are as described above.
Also, chaperone genes derived from other organism species may be codon-modified genes that are modified so as to improve translation efficiency via substitution of a nucleotide sequence with a codon that is frequently used in a host cell (the term “codon-modified” may be occasionally referred to as “synthesized” herein). DNA having a modified nucleotide sequence can be artificially synthesized. In the case of a long DNA sequence, a sequence is first divided into several fragments, fragments are synthesized in advance, and the resultants are then bound to each other at the end. Thus, DNA of interest can be synthesized.
In the present invention, one or more types of the aforementioned chaperone genes are used in combination. When two or more genes are used in combination, such genes may be derived from the same or different organism species.
Preferable examples of the chaperone genes that are used in the present invention include the PDI1 gene derived from O. minuta, the PDI1 gene derived from S. cerevisiae, the PDI gene derived from a human, the PDI gene derived from a human (a codon-modified gene), the ERO1 gene derived from O. minuta, the ERO1 gene derived from a human, and the Kar2 gene derived from O. minuta. 
Further preferable examples of the chaperone genes that are used in the present invention include a combination of the PDI1 gene and the ERO1 gene derived from O. minuta, a combination of the PDI1 gene and the Kar2 gene derived from O. minuta, a combination of the PDI gene derived from a human (a codon-modified gene) and the ERO1 gene derived from O. minuta, a combination of the PDI1 gene, the ERO1 gene, and the Kar2 gene derived from O. minuta, a combination of the PDI gene, the ERO1-Lβ gene, and the GRP78 gene derived from a human (codon-modified genes), and a combination of the PDI gene derived from a human (a codon-modified gene), the ERO gene derived from O. minuta, and the GRP78 gene derived from a human (a codon-modified gene).
The term “codon-modified gene” used with regard to the chaperone gene derived from a human refers to a gene in which frequency of a codon to be used is optimized in accordance with that of O. minuta. 
In the present invention, the aforementioned genes used for high-level secretory production of proteins and genes encoding foreign proteins that are the targets 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 20 mer to 30 mer 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 a single type of the chaperone gene, a vector comprising two or more copies of a single type of the chaperone genes, or a vector comprising a combination of two or more of the chaperone genes. In order to express the chaperone gene in a host cell, a vector comprising either gene may be used to carry out transformation. Alternatively, a vector comprising a plurality of 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 lysozyme gene, the α-amylase gene, and the α-galactosidase gene, and in particular, glycosyltransferase genes that are necessary for production of pharmaceutically useful glycoproteins, such as the erythropoietin (EPO) gene and the granulocyte colony-stimulating factor (G-CSF) genes; various interferon genes that are and physiologically active proteins, such as interferon α and interferon γ genes; various interleukin genes, such as IL1 and IL2 genes; 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 chaperone 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 promoter, dihydroxyacetone synthase (DAS) gene promoter, and formate dehydrogenase (FDH) gene promoter. 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, aggregated, 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.
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, Arg1, Arg2, Arg3, 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, 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 (MATa Δhis3Δleu2Δmet15Δ ura3) can be used as the Saccharomyces cerevisiae strains, although the yeast strains are not limited thereto.
Further, the present invention is intended to obtain a host cell in which the endoplasmic reticulum (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 fungus bodies, or destroyed cells or fungus bodies, 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 fungus bodies or cells after culture, the fungus bodies 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 fungus body or cells, the culture solution is used as it is, or the fungus body 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.
In the present invention, when yeast is used as a host cell, the aforementioned culture is more 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 O sugar chain can be inhibited. This also accelerates protein assembly 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 chaperone 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). PMT is important for generation of mannoproteins that constitute the yeast cell 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. Deficiency of the PMT gene may be single or double deficiency. As described above, PMT is an important gene for the growth of yeast. When activity, such as PMT gene deficiency, is eliminated or extremely lowered, the cell wall becomes fragile. Thus, the use of a PMT gene-deficient strain requires attention. A strain in which the PMT gene has been destroyed is preferably a strain in which either the PMT5 or PMT6 gene has been destroyed or a strain in which both the PMT5 gene and the PMT6 gene have been destroyed.
Examples of methods for suppressing the PMT gene include a method involving the use of antisense RNA or RNAi and a method involving attenuating a promoter.
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 Vector for Antibody Gene Introduction
(1) Construction of Vector for Antibody Gene Introduction Carrying a Zeocin-Resistant Gene as a Selection Marker and Comprising the Gap Gene Promoter and the Terminator Cassette
In order to prepare a Zeocin-resistant gene marker, a synthesized DNA fragment as shown in SEQ ID NO: 15 was subjected to double digestion with HindIII and KpnI restriction enzymes to obtain a DNA fragment containing a Zeocin-resistant gene.
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. Thus, the pOMexGP1UΔSp plasmid was obtained. The resulting pOMexGP1UΔSp was subjected to double digestion with HindIII and KpnI restriction enzymes to isolate a fragment containing the GAP gene promoter and the terminator, and a DNA fragment containing the Zeocin-resistant gene marker was introduced therein to obtain the pOMexGP1Z plasmid.
After the 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 ends of the heavy chain gene of the anti-TRAIL receptor antibody gene (WO 2001/083560, synthesized by Takara Bio while taking the frequency of the use of codons of O. minuta into consideration), the resultant was digested with XbaI and BamHI to obtain a fragment of the antibody heavy chain gene.
The resulting artificially synthesized antibody heavy chain gene was used as a template to carry out PCR using the SynCH-F primer and the SynCH-R primer shown below 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 constant region of the antibody heavy chain gene was obtained.









(SEQ ID NO: 16)



SynCH-F:



5′-GGAGCTCAAAAAGCTAGCACCAAGGGTCCATCCGTTTTCC-3′



 



(SEQ ID NO: 17)



SynCH-R:



5′-CAGATCTTTACTTACCTGGGGACAAGGACAAG-3′






The resulting DNA fragment was subjected to double digestion with the SacI and BglII restriction enzymes, and the resultant was introduced into pOMexGP1Z, which had been subjected to double digestion with the SacI and BamHI restriction enzymes. The resulting vector was designated as pOMexGPZ/SynCH. A variable region of the antibody heavy chain gene can be introduced into a site between the SacI and the NheI restriction enzyme sites of this vector.
(2) Construction of a Vector for Antibody Gene Introduction Carrying the ADE1 Gene as a Selection Marker and Comprising the Gap Gene Promoter and the Terminator Cassette
pOMexGP1U disclosed in WO 2003/091431 was treated with EcoT22I and blunt-ended. The BamHI linker was introduced therein to obtain pOMexGP2U. pOMexGP2U was treated with SalI and blunt-ended. The SpeI linker was introduced therein to obtain pOMexGP3U. pOMexGP3U was digested with HindIII and KpnI to isolate a fragment of approximately 2.0 kb comprising the GAP expression cassette. The resulting fragment was ligated to a fragment of approximately 5.0 kb comprising the ADE1 marker isolated by treating pOMex4A (disclosed in WO 2003/091431) with HindIII and KpnI to obtain pOMexGP1A. The fragment obtained by cleaving pOMexGP1A with the BsiWI restriction enzyme was blunt-ended, and the NdeI linker (Takara Bio) was introduced. The resulting plasmid was designated as pOMexGP2A.
After the 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 ends of the light chain gene of the anti-TRAIL receptor antibody gene (WO 2001/083560, synthesized by Takara Bio while taking the frequency of the use of codons of O. minuta into consideration), the resultant was digested with XbaI and BamHI to obtain a fragment of the antibody light chain gene.
The resulting artificially synthesized antibody light chain gene was used as a template to carry out PCR using the SynCL-F primer and the SynCL-R primer shown below 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 constant region of the antibody light chain gene was obtained.









(SEQ ID NO: 18)



SynCL-F primer:



5′-GACTAGTAAAAACGTACGGTTGCTGCTCCATCCGTTTTCAT



C-3′



 



(SEQ ID NO: 19)



SynCL-R primer:



5′-CAGATCTTTAGCACTCACCTCTGTTGAAGGAC-3′






The resulting DNA fragment was subjected to double digestion with the SpeI and BglII restriction enzymes, and the resultant was introduced into pOMexGP2A, which had been subjected to double digestion with the SpeI and BamHI restriction enzymes. The resulting vector was designated as pOMexGPA/SynCL. A variable region of the antibody light chain gene can be introduced into a site between the SpeI and the BsiWI restriction enzymes of this vector.
Example 2
Construction of Expression Vector for Antibody Gene
In order to express the Kar2 signal derived from O. minuta (hereafter refers to as the “OmKar2 signal”) and the light chain and the heavy chain of the anti-TRAIL receptor antibody as a fusion protein, the OmKar2 signal sequence was ligated to the anti-TRAIL receptor antibody gene (WO 2001/083560, synthesized by Takara Bio while taking the frequency of the use of codons of O. minuta into consideration) via overlap extension PCR using the oligonucleotide primers below.







For OmKar2 signal and heavy chain of anti-TRAIL


receptor antibody


(SEQ ID NO: 20)


Om-Kar2-Sac:


5′-GGAGCTCATGTTTAAGTTCAACCGCTC-3′


 


(SEQ ID NO: 21)


OmKar-SanH-R3:


5′-CAACGAGTTGAACCTCCGCCTCTGCTTCCACG-3′


 


(SEQ ID NO: 22)


OmKar-SanH-F3:


5′-CGTGGAAGCAGAGGCGGAGGTTCAACTCGTTG-3′


 


(SEQ ID NO: 23)


SanH-Nhe:


5′-GGCTAGCGGAGGAAACGGTAAC-3′


 


For OmKar2 signal and light chain of anti-TRAIL


receptor antibody


(SEQ ID NO: 24)


Om-Kar2-Spe:


5′-GACTAGTATGTTTAAGTTCAACCGCTC-3′


 


(SEQ ID NO: 25)


OmKar-SanL-R3:


5′-GGGTCATCTGGATGTCCGCCTCTGCTTCCACG-3′


 


(SEQ ID NO: 26)


OmKar-SanL-F3:


5′-CGTGGAAGCAGAGGCGGACATCCAGATGACCC-3′


 


(SEQ ID NO: 27)


SanL-Bsi:


5′-GCGTACGCTTGATCTCAACC-3′






The region of the OmKar2 signal sequence was amplified using genomic DNA of O. minuta prepared using the Y-DER Yeast DNA Extraction Reagent (PIERCE, 78870) as a template. With the use of AccuPrime Pfx DNA Polymerase (Invitrogen, 12344-024), PCR was carried out using the Om-Kar2-Sac and OmKar-SanH-R3 primers for the heavy chain and the Om-Kar2-Spe and OmKar-SanL-R3 primers for the light chain at 95° C. for 10 seconds, 55° C. for 30 seconds, and 68° C. for 30 seconds, and this cycle was repeated 30 times. Thus, the amplified target DNA fragments of about 0.1 kb were recovered.
The antibody gene region was amplified using DNA obtained by synthesizing the codon of the anti-TRAIL receptor antibody cDNA (WO 2001/083560) in accordance with the frequency of the codon of O. minuta used as a template. With the use of AccuPrime Pfx DNA Polymerase (Invitrogen, 12344-024), PCR was carried out using the OmKar-SanH-F3 and SanH-Nhe primers for the heavy chain and the OmKar-SanL-F3 and SanL-Bsi primers for the light chain at 95° C. for 10 seconds, 55° C. for 30 seconds, 68° C. for 30 seconds, and this cycle was repeated 30 times. The amplified target DNA fragment of the heavy chain variable region of approximately 0.36 kb and that of the light chain variable region of approximately 0.33 kb were recovered.
Subsequently, the amplified OmKar2 signal region for the heavy chain and the heavy chain variable region were used as templates, PCR was carried out using the Om-Kar2-Sac and SanH-Nhe primers and AccuPrime Pfx DNA Polymerase (Invitrogen, 12344-024) at 95° C. for 10 seconds, 55° C. for 30 seconds, and 68° C. for 30 seconds, and this cycle was repeated 30 times. Thus, an amplified target DNA fragment of approximately 0.47 kb was recovered. Also, the amplified OmKar2 signal region for the light chain and the light chain variable region were used as templates, and PCR was carried out using the Om-Kar2-Spe and SanL-Bsi primers and AccuPrime Pfx DNA Polymerase (Invitrogen, 12344-024) at 95° C. for 10 seconds, 55° C. for 30 seconds, and 68° C. for 30 seconds, and this cycle was repeated 30 times. Thus, an amplified target DNA fragment of approximately 0.43 kb was recovered. The recovered DNA fragments were cloned into pCR2.1-TOPO. Based on the nucleotide sequences of the inserted DNA fragments, the OmKar2 signal-antibody heavy chain variable region and the OmKar2 signal-antibody light chain variable region were confirmed to have genes that are fused in frame to each other. From a plasmid having a DNA fragment, the nucleotide sequence thereof had been confirmed, a DNA fragment containing the OmKar signal-antibody heavy chain variable region was recovered via digestion with SacI and NheI with the use of the SacI restriction enzyme site introduced into the Om-Kar2-Sac primer and the NheI restriction enzyme site introduced into the SanH-Nhe primer. Separately, the SpeI restriction enzyme site introduced into the Om-Kar2-Spe primer and the BsiWI restriction enzyme site introduced into the SanL-Bsi primer were used to recover a DNA fragment containing the OmKar signal-antibody light chain variable regions via digestion with SpeI and BsiWI.
In order to express the antibody heavy chain and the antibody light chain in O. minuta, a DNA fragment encoding the OmKar signal-antibody heavy chain variable region recovered via double digestion with SacI and NheI restriction enzymes and a DNA fragment encoding the OmKar signal-antibody light chain variable region recovered via double digestion with SpeI and BsiWI restriction enzymes were ligated to the expression vector pOMexGPZ/SynCH for the human IgG1γ-chain constant region obtained via double digestion with SacI and NheI restriction enzymes (prepared in Example 1) and the expression vector pOMexGPA/SynCL for the human IgG1κ-chain constant region obtained via double digestion with SpeI and BsiWI restriction enzymes (prepared in Example 1), respectively. The resulting plasmids were designated as onaP02706 and onaP03106.
Example 3
Construction of Expression Vector for Chaperone Gene Alone
(1) Construction of Constitutive Expression Vector for OmPDI1 Gene
The gene consisiting of the nucleotide sequence as shown in SEQ ID NO: 1 (1551 bp) is deduced to encode a protein consisiting of the amino acid sequence as shown in SEQ ID NO: 2 (516 amino acid residues).
The above protein has nucleotide sequence homology of about 60.5% to PDI1 (YCL043C) of S. cerevisiae and putative amino acid sequence homology of about 46.9%. The protein was deduced to be a functional homolog that also comprises two thioredoxin-like domains, CGHC (Cys-Gly-His-Cys), in PDI1 of S. cerevisiae, and it was designated as OmPDI1 as PDI1 of O. minuta. Since the nucleotide sequence of OmPDI1 comprises the cleavage sites for EcoT22I and SalI restriction enzymes, the EcoT22I and SalI restriction enzyme sites were modified via overlap extension PCR using the oligonucleotide primers below.









(SEQ ID NO: 28)










OMPDI1SAL:
5′-GGTCGACATGAAGTTATTTGGATTGAC-3′



 









(SEQ ID NO: 29)










OMPDI1T22I:
5′-GATGCATTTACAACTCGTCGTGAGCCAC-3′



 









(SEQ ID NO: 30)










OMPDI912F:
5′-GAGATACGGTATGCACGCCAAGAAC-3′



 









(SEQ ID NO: 31)










OMPDI936R:
5′-GTTCTTGGCGTGCATACCGTATCTC-3′



 









(SEQ ID NO: 32)










OMPDI1321F:
5′-GTTGCCGGTGTTGACATCGCCGG-3′



 









(SEQ ID NO: 33)










OMPDI1343R:
5′-CCGGCGATGTCAACACCGGCAAC-3′






PCR was carried out using the genomic DNA of O. minuta prepared using the Y-DER yeast DNA extraction reagent (78870, PIERCE) as a template, the OMPDI1SAL and the OMPDI936R primers, the OMPDI912F and the OMPDI1343R primers, and the OMPDI1321F and the OMPDI1T22I primers, and AccuPrime Pfx DNA Polymerase (Invitrogen, 12344-024) at 95° C. for 10 seconds, 55° C. for 30 seconds, and 68° C. for 60 seconds, and this cycle was repeated 30 times. Thus, target DNA fragments of approximately 0.94 kb, approximately 0.43 kb, and approximately 0.23 kb were amplified. Subsequently, PCR was carried out using the amplified fragments above and the OMPDI1SAL and the OMPDI1T22I primers 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 further amplified, and the resultant was cloned into pCR2.1-TOPO. Based on the nucleotide sequence of the inserted DNA fragment, it was confirmed that the target fragment had the OmPDI1 gene. From a plasmid carrying the DNA fragment with a confirmed nucleotide sequence, a DNA fragment containing OmPDI1 was recovered via digestion with SalI and EcoT22I, with the use of the SalI restriction enzyme site introduced into the OMPDI1SAL primer and the EcoT22I restriction enzyme site introduced into the OMPDI1T22I primer. In order to constitutively express OmPDI1 in O. minuta, OmPDI1 was ligated to pOMexGP1U disclosed in WO 2003/091431 recovered via digestion with SalI and EcoT22I. The resulting plasmid was designated as onaP03606.
(2) Construction of Constitutive Expression Vector for OmMPD1 Gene
The gene consisiting of the nucleotide sequence as shown in SEQ ID NO: 3 (936 bp) is deduced to encode a protein consisiting of the amino acid sequence as shown in SEQ ID NO: 4 (311 amino acid residues).
The above protein has nucleotide sequence homology of about 47.3% to MPD1 (YOR288C) of S. cerevisiae and putative amino acid sequence homology of about 31.2%. The protein was deduced to be a functional homolog that also comprises one thioredoxin-like domain, CGHC (Cys-Gly-His-Cys), contained in MPD1 derived from S. cerevisiae, and it was designated as OmMPD1 as MPD1 derived from O. minuta. The resultant was amplified via PCR using the oligonucleotide primers below.









(SEQ ID NO: 34)










OMIC1379SAL:
5′-GGTCGACATGAAAGTGGCAAGTTTG-3′



 









(SEQ ID NO: 35)










OMIC1379T22I:
5′-GATGCATTCATAGCTCATCTTTTTC-3′






PCR was carried out using the genomic DNA of O. minuta prepared using the Y-DER yeast DNA extraction reagent (78870, PIERCE) as a template and AccuPrime Pfx DNA Polymerase (Invitrogen, 12344-024) at 95° C. for 10 seconds, 55° C. for 30 seconds, and 68° C. for 60 seconds, and this cycle was repeated 30 times. Thus, a target DNA fragment of approximately 0.94 kb was amplified, and the resultant was into pCR2.1-TOPO. Based on the nucleotide sequence of the inserted DNA fragment, it was confirmed that the target fragment had the OmMPD1 gene. From a plasmid carrying the DNA fragment with a confirmed nucleotide sequence, a DNA fragment containing OmMPD1 was recovered via digestion with SalI and EcoT22I, with the use of the SalI restriction enzyme site introduced into the OMIC1379SAL primer and the EcoT22I restriction enzyme site introduced into the OMIC1379T22I primer. In order to constitutively express OmMPD1 in O. minuta, OmMPD1 was ligated to pOMexGP1U disclosed in WO 2003/091431 recovered via digestion with SalI and EcoT22I. The resulting plasmid was designated as onaP04006.
(3) Construction of Constitutive Expression Vector for OmSCJ1 Gene
The gene consisiting of the nucleotide sequence as shown in SEQ ID NO: 5 (930 bp) is deduced to encode a protein consisiting of the amino acid sequence as shown in SEQ ID NO: 6 (309 amino acid residues).
The above protein has nucleotide sequence homology of about 54.8% to SCJ1 (YMR214W) of S. cerevisiae and putative amino acid sequence homology of about 36.6%. The protein was deduced to be a functional homolog that comprises the central cysteine-rich (CR) domain of the DnaJ protein of CXXCXGXG and a region partially homologous to the DnaJ-C terminal region, and it was designated as OmSCJ1 as SCJ1 of O. minuta. The resultant was amplified via PCR using the oligonucleotide primers below.







(SEQ ID NO: 36)








OMSCJ1SAL:
5′-GGTCGACATGTTTATGGAGATCGGAG-3′


 







(SEQ ID NO: 37)








OMSCJ1T22I:
5′-GATGCATTCACAGCTCGTCGTGCAAC-3′






PCR was carried out using the genomic DNA of O. minuta prepared using the Y-DER yeast DNA extraction reagent (78870, PIERCE) as a template and AccuPrime Pfx DNA Polymerase (Invitrogen, 12344-024) at 95° C. for 10 seconds, 55° C. for 30 seconds, and 68° C. for 60 seconds, and this cycle was repeated 30 times. Thus, a target DNA fragment of approximately 0.93 kb was amplified, and the resultant was cloned into pCR2.1-TOPO. Based on the nucleotide sequence of the inserted DNA fragment, it was confirmed that the target fragment had the OmSCJ1 gene. From a plasmid carrying the DNA fragment with a confirmed nucleotide sequence, a DNA fragment containing OmSCJ1 was recovered via digestion with SalI and EcoT22I, with the use of the SalI restriction enzyme site introduced into the OMSCJ1SAL primer and the EcoT22I restriction enzyme site introduced into the OMSCJ1T22I primer. In order to constitutively express OmSCJ1 in O. minuta, OmSCJ1 was ligated to pOMexGP1U disclosed in WO 2003/091431 recovered via digestion with SalI and EcoT22I. The resulting plasmid was designated as onaP03806.
(4) Construction of Constitutive Expression Vector for OmEUG1 Gene
The gene consisiting of the nucleotide sequence as shown in SEQ ID NO: 7 (1137 bp) is deduced to encode a protein consisiting of the amino acid sequence as shown in SEQ ID NO: 8 (378 amino acid residues).
Although the above protein did not show obvious homology to the protein disulfide isomerase family of S. cerevisiae, the protein comprised thioredoxin-like domains of CHSC in a region comprising residues 52 to 55 and of CGYC in a region comprising residues 174 to 177 from the N terminus. Five types of protein disulfide isomerases having thioredoxin-like domains are known in S. cerevisiae; however, those having two thioredoxin-like domains in the molecules are limited to PDI1 and EUG1. Since P5 that belongs to the human PDI family is the only isomerase having two thioredoxin-like domains from the N-terminus to the central region of the molecule (Gene, 1995, 164, pp. 377-378), the above protein is deduced to function as a protein disulfide isomerase. Also, the protein was deduced to have the ER retention domain of KDEL at the C terminus and to function as protein disulfide isomerase in the yeast endoplasmic reticulum.
Accordingly, the above protein was deduced to be a functional homolog of EUG1 derived from S. cerevisiae, and it was designated as OmEUG1 as EUG1 of O. minuta. Since the nucleotide sequence of OmEUG1 comprises cleavage sites for EcoT22I and SalI restriction enzymes, the EcoT22I and the SalI restriction enzyme sites were modified via overlap extension PCR using the oligonucleotide primers below.









(SEQ ID NO: 38)










OMEUG1SAL:
5′-GGTCGACATGAAAGTCACGTCTATCTGG-3′



 









(SEQ ID NO: 39)










OMEUG1T22I:
5′-GATGCATTCACAGCTCATCCTTGGCTGG-3′



 









(SEQ ID NO: 40)










OMEUG819F:
5′-GATACACGCAGTTGACGAGCTG-3′



 









(SEQ ID NO: 41)










OMEUG840R:
5′-CAGCTCGTCAACTGCGTGTATC-3′



 









(SEQ ID NO: 42)










OMEUG925F:
5′-GACAACGCATTGTCGAAAGAAG-3′



 









(SEQ ID NO: 43)










OMEUG946R:
5′-CTTCTTTCGACAATGCGTTGTC-3′






PCR was carried out using the genomic DNA of O. minuta prepared using the Y-DER yeast DNA extraction reagent (78870, PIERCE) as a template, the OMEUG1SAL and the OMEUG840R primers, the OMEUG819F and the OMEUG946R primers, and the OMEUG925F and the OMEUG1T22I primers, and AccuPrime Pfx DNA Polymerase (Invitrogen, 12344-024) at 95° C. for 10 seconds, 55° C. for 30 seconds, and 68° C. for 60 seconds, and this cycle was repeated 30 times. Thus, target DNA fragments of approximately 0.84 kb, approximately 0.13 kb, and approximately 0.21 kb were amplified. Subsequently, PCR was carried out using the above amplified fragments, the OMEUG1SAL and the OMEUG1T22I primers, and AccuPrime PfxDNA Polymerase (Invitrogen, 12344-024) at 95° C. for 10 seconds, 55° C. for 30 seconds, and 68° C. for 90 seconds, and this cycle was repeated 30 times. Thus, a target DNA fragment of approximately 1.1 kb was amplified and the resultant was cloned into pCR2.1-TOPO. Based on the nucleotide sequence of the inserted DNA fragment, it was confirmed that the target fragment had the OmEUG1 gene. From a plasmid carrying the DNA fragment with a confirmed nucleotide sequence, a DNA fragment containing OmEUG1 was recovered via digestion with SalI and EcoT22I with the utilization of the SalI restriction enzyme site introduced into the OMEUG1SAL primer and the EcoT22I restriction enzyme site introduced into the OMEUG1T22I primer. In order to constitutively express OmEUG1 in O. minuta, OmEUG1 was ligated to pOMexGP1U digested with SalI and EcoT22I disclosed in WO 2003/091431. The resulting plasmid was designated as onaP03706.
(5) Construction of Constitutive Expression Vector for OmERO1 Gene
The gene consisiting of the nucleotide sequence as shown in SEQ ID NO: 9 (1728 bp) is deduced to encode a protein consisiting of the amino acid sequence as shown in SEQ ID NO: 10 (575 amino acid residues).
The above protein has nucleotide sequence homology of about 37.4% to ERO1 (YML130C) of S. cerevisiae and putative amino acid sequence homology of about 35.1%. It is known that ERO1 of S. cerevisiae comprises 14 Cys residues and at least 10 Cys residues are associated with disulfide bond (Cell, 2007, 129, pp. 333-344). In particular, it is demonstrated that 4 Cys residues are important for ERO1 activity, Cys100-Cys105 is referred to as “shuttle disulfide” and associated with oxidation of PDI1, and Cys352-Cys355 is associated with reoxidation of reduced shuttle disulfide (Moll. Cell, 1999, 4, pp. 469-477). Homology search between S. cerevisiae ERO1 and SEQ ID NO: 10 demonstrates that Cys125-Cys130, which is equivalent to shuttle Cys of S. cerevisiae Cys100-Cys105, and Cys384-Cys387, which is equivalent to active Cys of S. cerevisiae Cys352-Cys355, are conserved. Thus, the above protein is deduced to have endoplasmic oxidoreductase-like activity. Thus, the above protein is deduced to be a functional homolog of S. cerevisiae ERO1, and it was designated as OmERO1 as ERO1 of O. minuta. Since the OmERO1 nucleotide sequence comprises a cleavage site for the SalI restriction enzyme, the SalI restriction enzyme site was modified via overlap extension PCR using the oligonucleotide primers below.







(SEQ ID NO: 44)








OMEROSAL:
5′-GGTCGACATGAAGCACGTGATAAGTGGC-3′


 







(SEQ ID NO: 45)








OMEROT22I:
5′-GATGCATTTATAGCTCCAAACGATACAG-3′


 







(SEQ ID NO: 46)








OMERO166F:
5′-GAGTTTGAGTCCACGCCTTTCCGCG-3′


 







(SEQ ID NO: 47)








OMERO190R:
5′-CGCGGAAAGGCGTGGACTCAAACTC-3′






PCR was carried out using the genomic DNA of O. minuta prepared using the Y-DER yeast DNA extraction reagent (78870, PIERCE) as a template, the OMEROSAL and OMERO190R primers and the OMERO166F and OMEROT22I primers, and AccuPrime Pfx DNA Polymerase (Invitrogen, 12344-024) at 95° C. for 10 seconds, 55° C. for 30 seconds, and 68° C. for 60 seconds, and this cycle was repeated 30 times. Thus, target DNA fragments of approximately 0.19 kb and approximately 1.56 kb were amplified. Subsequently, PCR was carried out using the above amplified fragments, the OMEROSAL and OMEROT22I primers, and AccuPrime PfxDNA Polymerase (Invitrogen, 12344-024) at 95° C. for 10 seconds, 55° C. for 30 seconds, and 68° C. for 120 seconds, and this cycle was repeated 30 times. Thus, a target DNA fragment of approximately 1.73 kb was amplified and the resultant was cloned into pCR2.1-TOPO. Based on the nucleotide sequence of the inserted DNA fragment, it was confirmed that the target fragment had the OmERO1 gene. From a plasmid carrying the DNA fragment with a confirmed nucleotide sequence, a DNA fragment containing OmERO1 was recovered via digestion with SalI and EcoT22I with the utilization of the SalI restriction enzyme site introduced into the OMEROSAL primer and the EcoT22I restriction enzyme site introduced into the OMEROT22I primer. In order to constitutively express OmERO1 in O. minuta, OmERO1 was ligated to pOMexGP1U recovered via digestion with SalI and EcoT22I disclosed in WO 2003/091431. The resulting plasmid was designated as onaP03906.
(6) Construction of Constitutive Expression Vector for OmHSP104 Gene
The gene consisiting of the nucleotide sequence as shown in SEQ ID NO: 11 (2700 bp) is deduced to encode a protein consisiting of the amino acid sequence as shown in SEQ ID NO: 12 (899 amino acid residues).
The above protein has nucleotide sequence homology of about 60.4% to HSP104 (YLL026W) of S. cerevisiae and putative amino acid sequence homology of about 63.4%. HSP104 of S. cerevisiae is a molecular chaperone that belongs to the HSP100/Clp family, and it cooperates with a molecular chaperone that belongs to the HSP70/HSP40 family and an auxiliary chaperone thereof to regenerate an aggregated protein. The above protein has two domains in its molecule; i.e., the Clp amino terminal domain and the AAA+domain, and it is deduced to have HSP104-like activity. Thus, the above protein was deduced to be a functional homolog of S. cerevisiae HSP104 and designated as OmHSP104 as HSP104 of O. minuta. 
PCR was carried out using the genomic DNA of O. minuta prepared using the Y-DER yeast DNA extraction reagent (78870, PIERCE) as a template, the OmHSP104salF primer (5′-GGTCGACATGGATTCTACGCAATTTAC-3′: SEQ ID NO: 48), the OmHSP104EcoTR primer (5′-GATGCATTTAATCGAGATCAGGACTGC-3′: SEQ ID NO: 49), and AccuPrime Pfx DNA Polymerase (Invitrogen, 12344-024) at 95° C. for 10 seconds, 55° C. for 30 seconds, and 68° C. for 180 seconds, and this cycle was repeated 30 times. Thus, a target DNA fragment of approximately 2.7 kb was amplified and the resultant was cloned into pCR2.1-TOPO. Based on the nucleotide sequence of the inserted DNA fragment, it was confirmed that the target fragment had the OmHSP104 gene. From a plasmid carrying the DNA fragment with a confirmed nucleotide sequence, a DNA fragment containing OmHSP104 was recovered via digestion with SalI and EcoT22I with the utilization of the SalI restriction enzyme site introduced into the OmHSP104salF primer and the EcoT22I restriction enzyme site introduced into the OmHSP104EcoTR primer. In order to constitutively express OmHSP104 in O. minuta, OmHSP104 was ligated to pOMexGP1U recovered via digestion with SalI and EcoT22I disclosed in WO 2003/091431. The resulting plasmid was designated as onaP07107.
(7) Construction of Constitutive Expression Vector for OmKar2 Gene
The gene consisiting of the nucleotide sequence as shown in SEQ ID NO: 13 (1998 bp) is deduced to encode a protein consisiting of the amino acid sequence as shown in SEQ ID NO: 14 (665 amino acid residues).
The above protein has nucleotide sequence homology of about 60.4% to Kar2 (YJL034W) of S. cerevisiae and putative amino acid sequence homology of about 74.6%. Thus, the protein of interest was deduced to be a functional homolog and designated as OmKar2 as Kar2 of O. minuta. The OmKar2 nucleotide sequence comprises cleavage sites for HindIII and KpnI restriction enzymes. In order to recover an expression cassette comprising a region from a promoter to a terminator after cloning into pOMEGPU-1, the HindIII and KpnI restriction enzyme sites were modified via overlap extension PCR using the oligonucleotide primers below.







(SEQ ID NO: 50)








OMKAR-F:
5′-GGTCGACATGTTTAAGTTCAACCGCTCTG-3′


 







(SEQ ID NO: 51)








OMKAR-R:
5′-GATGCATTCACAGCTCATCATGATCCCAG-3′


 







(SEQ ID NO: 52)








Karl-F:
5′-CACTAAGGATGCTGGAACCATTGCCGGTCTGGAAG-3′


 







(SEQ ID NO: 53)








Karl-R:
5′-CTTCCAGACCGGCAATGGTTCCAGCATCCTTAGTG-3′


 







(SEQ ID NO: 54)








Kar2-F:
5′-CCAGCCCCAAGAGGAACCCCACAAATTGAGGTGAC-3′


 







(SEQ ID NO: 55)








Kar2-R:
5′-GTCACCTCAATTTGTGGGGTTCCTCTTGGGGCTGG-3′


 







(SEQ ID NO: 56)








Kar3-F:
5′-CGGATTCGGCTCCAAACTTGATGAGGATGACAAGG-3′


 







(SEQ ID NO: 57)








Kar3-R:
5′-CCTTGTCATCCTCATCAAGTTTGGAGCCGAATCCG-3′






PCR was carried out using the genomic DNA of O. minuta prepared using the Y-DER yeast DNA extraction reagent (78870, PIERCE) as a template, the OMKAR-F and the Kar1-R primers, the Kar1-F and the Kar2-R primers, the Kar2-F and the Kar3-R primers, and the Kar3-F and the OMKAR-R primers, and AccuPrime Pfx DNA Polymerase (Invitrogen, 12344-024) at 95° C. for 10 seconds, 55° C. for 30 seconds, and 68° C. for 60 seconds, and this cycle was repeated 30 times. Thus, target DNA fragments of approximately 0.6 kb, approximately 0.95 kb, approximately 0.3 kb, and approximately 0.2 kb were amplified. Subsequently, PCR was carried out using the above amplified fragments, the OMKAR-F and the OMKAR-R primers, and AccuPrime PfxDNA Polymerase (Invitrogen, 12344-024) at 95° C. for 10 seconds, 55° C. for 30 seconds, and 68° C. for 90 seconds, and this cycle was repeated 30 times. Thus, a target DNA fragment of approximately 2.0 kb was amplified and the resultant was cloned into pCR2.1-TOPO. Based on the nucleotide sequence of the inserted DNA fragment, it was confirmed that the target fragment had the OmKar2 gene. From a plasmid carrying the DNA fragment with a confirmed nucleotide sequence, a DNA fragment containing OmKar2 was recovered via digestion with SalI and EcoT22I with the utilization of the SalI restriction enzyme site introduced into the OMKAR-F primer and the EcoT22I restriction enzyme site introduced into the OMKAR-R primer. In order to constitutively express OmKar2 in O. minuta, OmKar2 was ligated to pOMexGP1U recovered via digestion with SalI and EcoT22I disclosed in WO 2003/091431. The resulting plasmid was designated as onaP09007.
(8) Construction of Constitutive Expression Vector for ScPDI1 Gene
PDI1 (YCL043C) of S. cerevisiae consists of amino acid sequence (SEQ ID NO: 121) composed of 522 amino acid residues encoded by a 1569-bp nucleotide sequence (SEQ ID NO: 120). PCR was carried out using genomic DNA of S. cerevisiae prepared using the Y-DER Yeast DNA Extraction Reagent (PIERCE, 78870) as a template, the ScPDI-sal-F primer (5′-GGTCGACATGAAGTTTTCTGCTGGTG-3′: SEQ ID NO: 58), the ScPDI-EcoT-R primer (5′-GATGCATTTACAATTCATCGTGAATG-3′ SEQ ID NO: 59), and AccuPrime Pfx DNA Polymerase (Invitrogen, 12344-024) at 95° C. for 10 seconds, 55° C. for 30 seconds, and 68° C. for 90 seconds, and this cycle was repeated 30 times. Thus, a target DNA fragment of approximately 1.6 kb was amplified and the resultant was cloned into pCR2.1-TOPO. Based on the nucleotide sequence of the inserted DNA fragment, it was confirmed that the target fragment had the S. cerevisiae PDI1 (ScPDI1) gene. From a plasmid carrying the DNA fragment with a confirmed nucleotide sequence, a DNA fragment containing ScPDI1 was recovered via digestion with SalI and EcoT22I with the utilization of the SalI restriction enzyme site introduced into the ScPDI-sal-F primer and the EcoT22I restriction enzyme site introduced into the ScPDI-EcoT-R primer. In order to constitutively express ScPDI1 in O. minuta, ScPDI1 was ligated to pOMexGP1U recovered via digestion with SalI and EcoT22I disclosed in WO 2003/091431. The resulting plasmid was designated as onaP09307.
(9) Construction of Constitutive Expression Vector for Human PDI Gene
Human PDI (hPDI, Accession No. P07237) consists of amino acid sequence (SEQ ID NO: 141) composed of 508 amino acid residues encoded by a 1527-bp nucleotide sequence (SEQ ID NO: 140). Human full-length cDNA clone, AK095938, lacks a 20-bp DNA sequence (5′-GTACCTGCTGGTGGAGTTCT-3′) (SEQ ID NO: 169) from residues 126 to 145 from an initiation codon. A lacking 20-bp region was restored via overlap extension PCR using AK095938 as a template. Subsequently, PCR was carried out using the SalmodifiedPDI-F primer (5′-GGTCGACATGCTGCGCCGCGCTCTGC-3′: SEQ ID NO: 60), the EcoTmodifiedPDI-R primer (5′-GATGCATTTACAGTTCATCTTTCACAG-3′: SEQ ID NO: 61), and AccuPrime Pfx DNA Polymerase (Invitrogen, 12344-024) at 95° C. for 10 seconds, 55° C. for 30 seconds, and 68° C. for 90 seconds, and this cycle was repeated 30 times. Thus, a target DNA fragment of approximately 1.5 kb was amplified and the resultant was cloned into pCR2.1-TOPO. Based on the nucleotide sequence of the inserted DNA fragment, it was confirmed that the target fragment had the hPDI gene. From a plasmid carrying the DNA fragment with a confirmed nucleotide sequence, a DNA fragment containing hPDI was recovered via digestion with SalI and EcoT22I with the utilization of the SalI restriction enzyme site introduced into the SalmodifiedPDI-F primer and the EcoT22I restriction enzyme site introduced into the EcoTmodifiedPDI-R primer. In order to constitutively express hPDI in O. minuta, hPDI was ligated to pOMexGP1U recovered via digestion with SalI and EcoT22I disclosed in WO 2003/091431. The resulting plasmid was designated as onaP09207.
(10) Construction of Constitutive Expression Vector for Codon-Modified Human PDI Gene
Human PDI (hPDI, Accession No. P07237) consists of amino acid sequence composed of 508 amino acid residues encoded by a 1527-bp nucleotide sequence. The hPDI gene was synthesized by taking codon preference of O. minuta into consideration (Operon Biotechnologies). From a plasmid carrying the DNA fragment with a confirmed nucleotide sequence, a DNA fragment (SEQ ID NO: 142) containing synthesized hPDI was recovered via digestion with SalI and EcoT22I with the utilization of the SalI restriction enzyme site and the EcoT22I restriction enzyme site, which had been introduced at the time of synthesis. In order to constitutively express synthesized hPDI in O. minuta, synthesized hPDI was ligated to pOMexGP1U recovered via digestion with SalI and EcoT22I disclosed in WO 2003/091431. The resulting plasmid was designated as onaP09107.
Example 4
Construction of Expression Vector for Chaperone Gene Combination
(1) Construction of pZ/GpGt Vector for Second Chaperone Introduction
In order to coexpress two chaperone genes, a vector comprising the GAP promoter and the GAP terminator and carrying, as a selection marker, a Zeocin-resistant gene was constructed in the following manner. In order to substitute the cleavage site for the HindIII restriction enzyme located upstream of the GAP promoter and the cleavage site for the KpnI restriction enzyme located downstream of the GAP terminator of pOMexGP1U with the ApaI restriction enzyme recognition sequence, GGGCCC, PCR was carried out using the GAPp-02Apa primer (5′-GCAGGGCCCTACTGGTTCAAGG-3′: SEQ ID NO: 62), the GAPt-02Apa primer (5′-GCAGGGCCCGCTCGAATCGAC-3′: SEQ ID NO: 63), and AccuPrime Pfx DNA Polymerase (Invitrogen, 12344-024) at 95° C. for 10 seconds, 55° C. for 30 seconds, and 68° C. for 120 seconds, and this cycle was repeated 30 times. The resulting target DNA fragment of approximately 2.1 kb was cloned into pCR2.1-TOPO. Based on the nucleotide sequence of the inserted DNA fragment, it was confirmed that the target fragment comprised the GAP promoter region and the GAP terminator region. The DNA fragment with a confirmed nucleotide sequence was introduced into the ApaI site of the pOMexGPZ/SynCH-AA vector, substituted with the ApaI restriction enzyme recognition sequence via insertion of the pApa I linker (Takara Bio, 4605P) into the HindIII restriction enzyme recognition sequence located upstream of the GAP promoter and the KpnI restriction enzyme recognition sequence located downstream of the GAP terminator of pOMexGPZ/SynCH. Thus, the pZ/GpGt vector comprising the GAP promoter and the GAP terminator that can be selected with the use of a Zeocin-resistant gene was constructed.
(2) Construction of Coexpression and Constitutive Expression Vector for OmPDI1 Gene and OmERO1 Gene
The OmERO1 region was recovered from the OmERO1 constitutive expression vector, onaP03906, with the aid of the SalI and EcoT22I restriction enzymes. After pZ/GpGt was digested with the SalI and EcoT22I restriction enzymes, an OmERO1-containing SalI-EcoT22I fragment was introduced to construct pZ/GpGt/OmERO1. Subsequently, pZ/GpGt/OmERO1 was digested with the ApaI restriction enzyme, and a fragment comprising the GAP promoter-OmERO1-GAP terminator (i.e., the OmERO1 expression cassette) was recovered. The recovered OmERO1 expression cassette was introduced into the ApaI restriction enzyme site of the OmPDI1 expression vector, onaP03606, the insertion direction was confirmed via PCR, and a vector into which the OmPDI1 and OmERO1 expression cassette was introduced in inverse orientation with respect to each other around the URA3 marker of the OmPDI1 expression vector, onaP03606, was selected. The resulting coexpression vector for OmPDI1 and OmERO1 was designated as onaP09507 (FIG. 1).
(3) Construction of Coexpression and Constitutive Expression Vector for OmPDI1 Gene and OmKar2 Gene
The OmKar2 region was recovered from the OmKar2 constitutive expression vector, onaP09007, with the aid of the SalI and EcoT22I restriction enzymes. After pZ/GpGt was digested with the SalI and EcoT22I restriction enzymes, an OmKar2-containing SalI-EcoT22I fragment was introduced to construct pZ/GpGt/OmKar2. Subsequently, pZ/GpGt/OmKar2 was digested with the ApaI restriction enzyme, and a fragment comprising the GAP promoter-OmKar2-GAP terminator (i.e., the OmKar2 expression cassette) was recovered. The recovered OmKar2 expression cassette was introduced into the ApaI restriction enzyme site of the OmPDI1 expression vector, onaP03606, the insertion direction was confirmed via PCR, and a vector into which the OmPDI1 and OmKar2 expression cassette was introduced in inverse orientation with respect to each other around the URA3 marker of the OmPDI1 expression vector, onaP03606, was selected. The resulting coexpression vector for OmPDI1 and OmKar2 was designated as onaP09707 (FIG. 1).
(4) Construction of Coexpression and Constitutive Expression Vector for OmPDI1 Gene and OmHSP104 Gene
The OmHSP104 region was recovered from the OmHSP104 constitutive expression vector, onaP07107, with the aid of the SalI and EcoT22I restriction enzymes. After pZ/GpGt was digested with the SalI and EcoT22I restriction enzymes, an OmHSP104-containing SalI-EcoT22I fragment was introduced to construct pZ/GpGt/OmHSP104. Subsequently, pZ/GpGt/OmHSP104 was digested with the ApaI restriction enzyme, and a fragment comprising the GAP promoter-OmHSP104-GAP terminator (i.e., the OmHSP104 expression cassette) was recovered. The recovered OmHSP104 expression cassette was introduced into the ApaI restriction enzyme site of the OmPDI1 expression vector, onaP03606, the insertion direction was confirmed via PCR, and a vector into which the OmPDI1 and OmHSP104 expression cassette was introduced in inverse orientation with respect to each other around the URA3 marker of the OmPDI1 expression vector, onaP03606, was selected. The resulting coexpression vector for OmPDI1 and OmHSP104 was designated as onaP09607 (FIG. 1).
(5) Construction of Constitutive Expression Vector Comprising Two Copies of OmPDI1 Gene Expression Cassettes
The OmPDI1 region was recovered from the OmPDI1 constitutive expression vector, onaP03606, with the aid of the SalI and EcoT22I restriction enzymes. After pZ/GpGt was digested with the SalI and EcoT22I restriction enzymes, an OmPDI1-containing SalI-EcoT22I fragment was introduced to construct pZ/GpGt/OmPDI1. Subsequently, pZ/GpGt/OmPDI1 was digested with the ApaI restriction enzyme, and a fragment comprising the GAP promoter-OmPDI1-GAP terminator (i.e., the OmPDI1 expression cassette) was recovered. The recovered OmPDI1 expression cassette was introduced into the ApaI restriction enzyme site of the OmPDI1 expression vector, onaP03606, the insertion direction was confirmed via PCR, and a vector into which an expression cassette of the first copy, OmPDI1, and an expression cassette of the second copy, OmPDI1, were introduced in inverse orientation with respect to each other around the URA3 marker of the OmPDI1 expression vector, onaP03606, was selected. The resulting coexpression vector comprising two copies of the OmPDI1 expression cassettes was designated as onaP09407 (FIG. 1).
(6) Construction of Coexpression and Constitutive Expression Vector for hPDI Gene and OmERO1 Gene
The OmERO1 region was recovered from the OmERO1 constitutive expression vector, onaP03906, with the aid of the SalI and EcoT22I restriction enzymes. After pZ/GpGt was digested with the SalI and EcoT22I restriction enzymes, an OmERO1-containing SalI-EcoT22I fragment was introduced to construct pZ/GpGt/OmERO1. Subsequently, pZ/GpGt/OmERO1 was digested with the ApaI restriction enzyme, and a fragment comprising the GAP promoter-OmERO1-GAP terminator (i.e., the OmERO1 expression cassette) was recovered. The recovered OmERO1 expression cassette was introduced into the ApaI restriction enzyme site of the constitutive expression vector for the codon-modified Human PDI gene, onaP09107, the insertion direction was confirmed via PCR, and a vector into which the codon-modified Human PDI and OmERO1 expression cassette was introduced in inverse orientation with respect to each other around the URA3 marker of the codon-modified Human PDI constitutive vector, onaP09107, was selected. The resulting coexpression vector for codon-modified Human PDI and OmERO1 was designated as onaP11107 (FIG. 2).
(7) Construction of Coexpression and Constitutive Expression Vector for OmPDI1, OmERO1, and OmKar2 Genes
A coexpression vector for three chaperone genes was constructed in the following manner.
(7-1) Construction of a Foreign Gene-Expressing Vector (pOMexPGHy) 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.
(i) Preparation of Probes
DNA degenerate primers comprising nucleotide sequences corresponding to the conserved amino acid sequences, i.e., RVDFNVPLD (SEQ ID NO: 170) and EGKELPGVA (SEQ ID NO: 171), derived from Saccharomyces cerevisiae (GenBank accession number: P00560) and Candida maltosa (GenBank accession number: P41757) were synthesized in the following manner.







(SEQ ID NO: 64)








PPG5:
5′-GN GTN GAY TTY AAY GTN CCN TTR GA-3′


 







(SEQ ID NO: 65)








PPG3:
5′-GY NAC DCC NGG YAA YTC YTT DCC YTC-3′






The PPG5 primer (SEQ ID NO: 64) corresponds to the amino acid sequence, RVDFNVPLD, and the PPG3 primer (SEQ ID NO: 65) is a sequence of a complementary strand of a nucleotide sequence corresponding to the amino acid sequence, EGKELPGVA. 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.
(ii) 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 (GE Healthcare Bio-Sciences). The DNA fragment obtained above was labeled with the use of the AlkPhos DIRECT (RPN3690, GE Healthcare Bio-Sciences), 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.
(iii) 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: 66. The nucleotide sequence as shown in SEQ ID NO: 66 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: 67 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%.
(iv) 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.







(SEQ ID NO: 68)


OPGK-P-F:


5′-AAGCTTGACAATGTAGGAGATCATAAACACATCGTGCGCGTC-3′


 


(SEQ ID NO: 69)


OPGK-P-R:


5′-GGATCCAGATCTCATATGACTAGTTGCTAGTTCTATGCGGCGTTAGT


GTTTACACTACGACAGCT-3′


 


(SEQ ID NO: 70)


OPGK-T-F:


5′-GGATCCGTGGGATTTGCGTGATCTACGTAGTGGTTATTTT-3′


 


(SEQ ID NO: 71)


OPGK-T-R:


5′-GGTACCGCAGTGAAAGGCGATGCCACCATGTGCAAGGAGTTC-3′






Using pOMPGK1 above as a template, PCR was carried out using the OPGK-P-F primer (SEQ ID NO: 68) and the OPGK-P-R primer (SEQ ID NO: 69) 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: 70) and the OPGK-T-R primer (SEQ ID NO: 71) 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.
(7-2) Construction of Coexpression Vector for OmPDI1 and OmERO1 Via Expression Regulation by GAP Promoter and for OmKar2 Via Expression Regulation by PGK Promoter
The PGK promoter and PGK terminator regions were cloned via PCR from the pOMexPGHy vector prepared in the above-described manner. The PGK promoter region was subjected to PCR using the OmPGKp-01Hd primer (5′-GGAAGCTTGACAATGTAGGAGATCATAAACA-3′: SEQ ID NO: 72) and the OmPGKp-02Sal primer (5′-GGTCGACTGCTAGTTCTATGCGGC-3′: SEQ ID NO: 73) and the PGK terminator region was subjected to PCR using the OmPGKt-01EcoT primer (5′-GGATGCATGTGGGATTTGCGTGATCTAC-3′: SEQ ID NO: 74) and the OmPGKt-02Kpn primer (5′-GGGTACCAGGGTCGATTTTCTTGGTCG-3′: SEQ ID NO: 75) at 95° C. for 10 seconds, 55° C. for 30 seconds, and 68° C. for 90 seconds with the use of AccuPrime Pfx DNA Polymerase (Invitrogen, 12344-024), and this cycle was repeated 30 times. The resulting target DNA fragments of approximately 1.5 kb and approximately 0.5 kb were cloned into pCR2.1-TOPO. Based on the nucleotide sequence of the inserted DNA fragment, it was confirmed that the target fragments comprised the PGK promoter and PGK terminator regions.
Subsequently, the constitutive expression vector for the OmKar2 gene, onaP09007, was digested with the EcoT22I and KpnI restriction enzymes and the resultant was ligated to the PGK terminator region recovered via digestion with the EcoT22I and KpnI restriction enzymes. Further, the resulting plasmid was digested with the HindIII and SalI restriction enzymes, and the resultant was ligated to the PGK terminator region recovered via digestion with the HindIII and SalI restriction enzymes. In the resulting plasmid, the GAP promoter and the GAP terminator were substituted with the PGK promoter and the PGK terminator, and OmKar2 expression was regulated by the PGK promoter. Thus, it was designated as the pOU1/Kar2Ppt vector (FIG. 3). The HindIII restriction enzyme site of pOU1/Kar2Ppt was blunt-ended, the cleavage site for the KpnI restriction enzyme was introduced with the use of the pKpnI linker (Takara Bio, 4668P), and the resulting vector was designated as pOU1/Kar2-PptK.
Subsequently, pOU1/Kar2-PptK was digested with the KpnI restriction enzyme, a fragment containing the PGK promoter-OmKar2-PGK terminator (i.e., the OmKar2 expression cassette) was recovered, and the resultant was introduced into the KpnI restriction enzyme site of the coexpression vector for OmPDI1 and OmERO1, onaP09507, to construct the coexpression vector for OmPDI1, OmERO1, and OmKar2, onaP11007 (FIG. 4).
Example 5
Preparation of Chaperone-Introduced Yeast Strain (O. minuta)
All the constitutive expression vectors for the chaperone gene constructed in Example 4 were digested with the NotI restriction enzyme and then introduced into the O. minuta YK5 strain (Δoch1Δyps1Δura3Δade1: a strain in which the Ogataea minuta protease YPS1 gene has been destroyed) via electroporation. Electroporation was carried out under the conditions described in WO 2003/091431. After electroporation, the resultant was applied on Casamino-U agar medium that had been sterilized via steaming under pressure (6.7 g/l of Yeast Nitrogen Base without amino acids, 0.5 g/l of casamino acid, 20 g/l of glucose, 20 mg/l of L-tryptophan, 20 mg/l of adenine, and 20 g/l of Bacto agar), followed by multiplication at 30° C. for approximately 2 to 3 days. The grown transformant was allowed to grow again on Casamino-U agar medium, a transformant into which the chaperone gene, expression of which is regulated by the GAP promoter, was selected via colony-PCR. Part of yeast that had been multiplied on Casamino-U agar medium was suspended in 10 μl of a 0.25% SDS solution, 90 μl of sterilized water was added, and yeast cells were then removed via centrifugation at 2,700×g and 4° C. for 5 minutes. The obtained supernatant was designated as a DNA solution. A strain that was confirmed to have been amplified with the use of the GAPpforS-F primer (5′-GATCTCAGGCCGAGTCAAGAC-3′: SEQ ID NO: 76) designed in the GAP promoter sequence and the primer shown below was designated as a strain into which a constitutive expression vector for the chaperone gene had been introduced. When constructing a coexpression vector for three chaperone genes, OmKar2 was expressed with the use of the PGK promoter. Thus, introduction of the PGK promoter-OmKar2-PGK terminator expression cassette was confirmed with the use of the PGKpforS-F primer (5′-TAACGCCGCATAGAACTAGC-3′: SEQ ID NO: 77) designed in the PGK promoter sequence and the OMKAR-R primer (5′-GATGCATTCACAGCTCATCATGATCCCAG-3′: SEQ ID NO: 51).
(p1) A primer used for confirming introduction of OmPDI1 and 2 copies of OmPDI1 OMPDI1T22I: 5′-GATGCATTTACAACTCGTCGTGAGCCAC-3′ (SEQ ID NO: 29)
(p2) A primer used for confirming introduction of OmMPD1 OMIC1379T22I: 5′-GATGCATTCATAGCTCATCTTTTTC-3′ (SEQ ID NO: 35)
(p3) A primer used for confirming introduction of OmSCJ1 OMSCJ1T22I: 5′-GATGCATTCACAGCTCGTCGTGCAAC-3′ (SEQ ID NO: 37)
(p4) A primer used for confirming introduction of OmEUG1 OMEUG1T22I: 5′-GATGCATTCACAGCTCATCCTTGGCTGG-3′ (SEQ ID NO: 39)
(p5) A primer used for confirming introduction of OmERO1 OMEROT22I: 5′-GATGCATTTATAGCTCCAAACGATACAG-3′ (SEQ ID NO: 45)
(p6) A primer used for confirming introduction of OmHSP104 OmHSP104EcoTR: 5′-GATGCATTTAATCGAGATCAGGACTGC-3′ (SEQ ID NO: 49)
(p7) A primer used for confirming introduction of OmKar2 OMKAR-R: 5′-GATGCATTCACAGCTCATCATGATCCCAG-3′ (SEQ ID NO: 51)
(p8) A primer used for confirming introduction of ScPDI1 ScPDI-EcoT-R: 5′-GATGCATTTACAATTCATCGTGAATG-3′ (SEQ ID NO: 59)
(p9) A primer used for confirming introduction of hPDI EcoTmodifiedPDI-R: 5′-GATGCATTTACAGTTCATCTTTCACAG-3′ (SEQ ID NO: 61)
(p10) A primer used for confirming introduction of synthesized hPDI ShPDI-ttaR: 5′-GATGCATTTACAACTCGTCCTTAAC-3′ (SEQ ID NO: 78)
(p11) A primer used for confirming introduction of OmPDI1+OmERO1 OMPDI1T22I: 5′-GATGCATTTACAACTCGTCGTGAGCCAC-3′ (SEQ ID NO: 29) and OMEROT22I: 5′-GATGCATTTATAGCTCCAAACGATACAG-3′ (SEQ ID NO: 45)
(p12) A primer used for confirming introduction of OmPDI1+OmKar2 OMPDI1T22I: 5′-GATGCATTTACAACTCGTCGTGAGCCAC-3′ (SEQ ID NO: 29) and OMKAR-R: 5′-GATGCATTCACAGCTCATCATGATCCCAG-3′ (SEQ ID NO: 51)
(p13) A primer used for confirming introduction of OmPDI1+OmHSP104 OMPDI1T22I: 5′-GATGCATTTACAACTCGTCGTGAGCCAC-3′ (SEQ ID NO: 29) and OmHSP104EcoTR: 5′-GATGCATTTAATCGAGATCAGGACTGC-3′ (SEQ ID NO: 49)
(p14) A primer used for confirming introduction of synthesized hPDI+OmERO1 ShPDI-ttaR: 5′-GATGCATTTACAACTCGTCCTTAAC-3′ (SEQ ID NO: 78) and OMEROT22I: 5′-GATGCATTTATAGCTCCAAACGATACAG-3′ (SEQ ID NO: 45):
(p15) A primer used for confirming introduction of OmPDI1+OmERO1+OmKar2 OMPDI1T22I: 5′-GATGCATTTACAACTCGTCGTGAGCCAC-3′ (SEQ ID NO: 29), OMEROT22I: 5′-GATGCATTTATAGCTCCAAACGATACAG-3′ (SEQ ID NO: 45), and OMKAR-R: 5′-GATGCATTCACAGCTCATCATGATCCCAG-3′ (SEQ ID NO: 51)
A target fragment was amplified via PCR using TaKaRa LA TaqTM with GC Buffer (Takara Bio, RR02AG) at 94° C. for 30 seconds, 55° C. for 30 seconds, and 72° C. for 60 to 180 seconds, and this cycle was repeated 30 times. The transformants below, which were confirmed to have been amplified, were designated as strains constitutively expressing chaperone.
(E1) The strain constitutively expressing OmPDI1, the ona03306 strain
(E2) The strain constitutively expressing OmMPD1, the ona03406 strain
(E3) The strain constitutively expressing OmSCJ1, the ona03506 strain
(E4) The strain constitutively expressing OmEUG1, the ona03706 strain
(E5) The strain constitutively expressing OmERO1, the ona03606 strain
(E6) The strain constitutively expressing OmHSP104, the ona13407 strain
(E7) The strain constitutively expressing OmKar2, the ona23007 strain
(E8) The strain constitutively expressing ScPDI1, the ona26907 strain
(E9) The strain constitutively expressing hPDI, the ona31107 strain
(E10) The strain constitutively expressing synthetic hPDI, the ona30807 strain
(E11) The strain constitutively expressing OmPDI1+OmERO1, the ona27607 strain
(E12) The strain constitutively expressing OmPDI1+OmKar2, the ona30507 strain
(E13) The strain constitutively expressing OmPDI1+OmHSP104, the ona27707 strain
(E14) The strain constitutively expressing 2 copies of OmPDI1, the ona27207 strain
(E15) The strain constitutively expressing synthetic hPDI+OmERO1, the ona45007 strain
(E16) The strain constitutively expressing OmPDI1+OmERO1+OmKar2, the ona44607 strain
Example 6
Construction of Antibody-Producing Yeast Strain (O. minuta)
The expression vector for the anti-TRAIL receptor antibody gene (WO 2001/083560) constructed in Example 2 was introduced into the yeast strain (O. minuta) to prepare an antibody-producing yeast strain. As the yeast strain (O. minuta), the ona01206 strain prepared from O. minuta YK5 strain in the following manner was used.
When the O. minuta YK5 strain did not complement a ura3 deletion mutation, the growth capacity was lowered, and the initiation codon of the ura3 gene was deleted. In order to complement a ura3 deletion mutation, accordingly, homologous recombination was carried out using the OmURA3 fragment to complement a ura3 deletion mutation. After the O. minuta YK5 strain was transformed with the use of the OmURA3 fragment via electroporation, the transformant was applied on Casamino-U agar medium, which had been sterilized via steaming under pressure, and the transformant was allowed to grow at 30° C. for approximately 2 to 3 days. The grown transformant was allowed to grow again on Casamino-U agar medium, part of yeast that had grown on Casamino-U agar medium was suspended in 10 μl of a 0.25% SDS solution, 90 μl of sterilized water was added, and yeast cells were removed via centrifugation at 2,700×g and 4° C. for 5 minutes. The obtained supernatant was designated as a DNA solution. With the use of the OmURA3F primer (5′-ATGTCCTCGACTAAGACATACGC-3′: SEQ ID NO: 79) and the OmURA3R primer (5′-TCATGCGACACGACTCAAATAAG-3′: SEQ ID NO: 80), which had been designed at the initiation codon side and the termination codon side of the ura3 gene, respectively, a target fragment of approximately 0.8 kb was amplified using TaKaRa LA TaqTM with GC Buffer (Takara Bio, RR02AG) (94° C. for 30 seconds, 55° C. for 30 seconds, and 72° C. for 60 seconds, and this cycle was repeated 30 times). The transformant, which was confirmed to have been amplified, was designated as the ona01206 strain as a ura3 candidate strain.
Subsequently, the expression vector for the anti-TRAIL receptor antibody gene (WO 2001/083560) constructed in Example 2 was introduced into the ona01206 strain via electroporation. As an antibody heavy chain, 1 μg of onaP02706 digested with the Sse8387I restriction enzyme was used. As an antibody light chain, 1 μg of onaP03106 digested with the NotI restriction enzyme was used. After electroporation, the transformant was applied on Casamino-U-A agar medium to which ZeocinTM (Invitrogen, R250-01) had been added at a concentration of 100 μg/ml (6.7 g/l of Yeast Nitrogen Base without amino acids, 0.5 g/l of casamino acid, 20 g/l of glucose, 20 mg/l of L-tryptophan, and 20 g/l of Bacto agar), and the transformant was allowed to grow at 30° C. for approximately 2 to 3 days. The grown transformant was allowed to grow again on Casamino-U-A agar medium to which ZeocinTM (Invitrogen, R250-01) had been added at a concentration of 100 μg/ml, and the resultant was designated as an antibody secretory-producing strain. The antibody-producing strain was cultured in the following manner. With the use of 2× YP-P6-GG medium (the medium was prepared by dissolving 20 g of Difco yeast extract and 40 g of Bacto peptone in 900 ml of pure water, sterilizing the solution via steaming under pressure, and adding 100 ml of separately sterilized 10× phosphate buffer (pH 6.0) (1M KH2PO4, 0.15M (NH4)2SO4, 0.375N KOH), 10 ml of a separately sterilized 50% glucose solution, and 25 ml of separately sterilized 80% glycerine), 800 μl to 1000 μl of 2× YP-P6-GG medium was introduced into a 96-deep well plate (Greiner, 780271), the strains were sowed with the use of a toothpick, and the top of the plate was sealed with CO2-permeable plate sealers (Greiner, 676051). Culture was carried out at an agitation speed of 310 rpm, an amplitude of 50 mm, at 30° C. for 3 or 4 days. The yeast cells were removed from the culture solution via centrifugation at 2,700×g and 4° C. for 5 minutes to prepare a culture supernatant, and the resultant was designated as a secretory antibody sample.
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 secretory antibody 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, 04-10-20)) and the ABTS peroxidase substrate (KPL, 50-66-01). The selected antibody-producing yeast strain, ona02306, exhibited the capacity for secreting and producing an antibody at approximately 0.8 mg/l (FIG. 5).
Example 7
Effects of Inhibition of O-Sugar Chain Addition of Yeast-Producing Antibody Caused by the Addition of an Inhibitor of Protein Mannosyl Transferase (PMT) on Antibody Production
The antibody-producing yeast obtained in Example 6 was cultured under conditions in which formation of O-sugar chain via the addition of PMT inhibitors is inhibited (PMT inhibitors, 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, was added to the medium). With the use of 2× YP-P6-GG medium [(the medium was prepared by dissolving 20 g of Difco yeast extract and 40 g of Bacto peptone in 900 ml of pure water, sterilizing the solution via steaming under pressure, and adding 100 ml of separately sterilized 10× phosphate buffer (pH 6.0) (1M KH2PO4, 0.15M (NH4)2SO4, 0.375N KOH), 10 ml of a separately sterilized 50% glucose solution, and 25 ml of separately sterilized 80% glycerine), 800 μl of 2× YP-P6-GG medium was introduced into a 96-deep well plate (Greiner, 780271), the strains were sowed with the use of a toothpick, and the top of the plate was sealed with CO2-permeable plate sealers (Greiner, 676051). Control culture was carried out at an agitation speed of 310 rpm, an amplitude of 50 mm, at 30° C. for 2 days, 100 μl of 2× YP-P6-GG medium was added, and 100 μl of 2× YP-P6-GG medium was further added on day 3. In the case of culture involving single addition of a rhodanine-3-acetic acid derivative 1c, culture was carried out at an agitation speed of 310 rpm, an amplitude of 50 mm, at 30° C. for 2 days, 100 μl of 2× YP-P6-GG medium was added, and 100 μl of 2× YP-P6-GG medium containing 20 μM of 1c was further added on day 3. In the case of culture involving addition of a rhodanine-3-acetic acid derivative 1c twice, culture was carried out at an agitation speed of 310 rpm, an amplitude of 50 mm, at 30° C. for 2 days, 100 μl of 2× YP-P6-GG medium containing 20 μM of 1c was added, and 100 μl of 2× YP-P6-GG medium containing 20 μM of 1c was further added on day 3.
Secretion and production of an antibody were confirmed via sandwich ELISA or Western blotting. Yeast cells were removed from the culture product via centrifugation at 2,700×g and 4° C. for 5 minutes, and the resulting culture supernatant was designated as a sample of secreted antibody. 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, the sample of secreted antibody 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, 04-10-20)) and the ABTS peroxidase substrate (KPL, 50-66-01).
Western blotting was carried out as follows. After the protein was subjected to SDS-PAGE under reducing and non-reducing conditions, the separated protein was blotted to a PVDF membrane, and the antibody heavy chain and the light chain were detected using Anti-human IgG (γ-chain specific) (Sigma, I-3382) and Goat anti human kappa b&f affinity purified (Bethyl, A-80-115A) as the primary antibodies. Peroxidase conjugated affinity purified anti-goat IgG (H&L) (Rabbit) (Rockland, #605-4313) was used as the secondary antibody. Detection was carried out using ECL Advance Western blotting detection kit (GE, RPN2135).
As shown in FIG. 5, a control sample (the ona02306 strain) exhibited the amount of secretion and production of an antibody at 0.8 mg/l. With a single addition of PMT inhibitors, the amount of antibody production was increased to approximately 1.7 mg/l, and it was increased to approximately 1.9 mg/l; i.e., about 2.1 to about 2.4 times greater, via the addition of PMT inhibitors twice. That is, the amount of antibody production was increased by approximately 2.1 to approximately 2.4 times via the addition of PMT inhibitors (FIG. 5). Also, Western blot analysis demonstrated that, in control culture, a high-molecular-weight antibody heavy chain with a smaller mobility than the antibody heavy chain was detected via reducing electrophoresis by an antibody heavy chain-specific antibody, although the high-molecular-weight antibody heavy chain was decreased via the addition of PMT inhibitors to culture (FIG. 6: reducing SDS-PAGE/WB, antibody Hc and antibody Lc were simultaneously detected). When transformed yeast was to be cultured with the use of a 96-deep well plate (Greiner, 780271) with the addition of PMT inhibitors, accordingly, culture was carried out at 30° C. for 2 days, and culture was further carried out by adding 100 μl of 2× YP-P6-GG medium containing 20 μm of 1c and then 100 μl of 2× YP-P6-GG medium containing 20 μm of 1c 3 days later.
Example 8
Construction of Antibody-Producing Yeast Strain (O. minuta) that Constitutively Expresses Chaperone
The expression vector for the anti-TRAIL receptor antibody gene (WO 2001/083560) constructed in Example 2 was introduced into each of the strains constitutively expressing chaperone alone bred in Example 5 via electroporation. As an antibody heavy chain, 1 μg of onaP02706 digested with the Sse8387I restriction enzyme was used. As an antibody light chain, 1 μg of onaP03106 digested with the NotI restriction enzyme was used. After electroporation, the transformant was applied on Casamino-U-A agar medium to which ZeocinTM (Invitrogen, R250-01) had been added at a concentration of 100 μg/ml (6.7 g/l of Yeast Nitrogen Base without amino acids, 0.5 g/l of casamino acid, 20 g/l of glucose, 20 mg/l of L-tryptophan, and 20 g/l of Bacto agar), and the transformant was allowed to grow at 30° C. for approximately 2 to 3 days. The grown transformant was allowed to grow again on Casamino-U-A agar medium to which ZeocinTM (Invitrogen, R250-01) had been added at a concentration of 100 μg/ml and a strain that secretes and produces an antibody was screened for. The ona08906 strain was selected as the strain constitutively expressing OmSCJ1 and having the antibody gene introduced therein, the ona09206 strain was selected as the strain constitutively expressing OmEUG1 and having the antibody gene introduced therein, the ona09406 strain was selected as the strain constitutively expressing OmERO1 and having the antibody gene introduced therein, the ona26407 strain was selected as the strain constitutively expressing OmKar2 and having the antibody gene introduced therein, the ona09506 strain was selected as the strain constitutively expressing OmPDI1 and having the antibody gene introduced therein, and the ona15807 strain was selected as the strain constitutively expressing OmHSP104 and having the antibody gene introduced therein.
Example 9
Effects of Chaperone on Secretory Production of Antibody
With the use of the strain constitutively expressing chaperone and having the antibody gene introduced therein obtained in Example 8, the effects of chaperone introduction on secretory production of antibody were examined. Also, the effects of enhancing secretory production of antibody caused by the addition of PMT inhibitors that were found in Example 7 were examined. As shown in FIG. 7, a control strain (i.e., the ona02306 strain) exhibited the amount of antibody secretory production of approximately 0.8 mg/l, the strain constitutively expressing OmSCJ1 and having the antibody gene introduced therein (i.e., the ona08906 strain) exhibited that of approximately 2.6 mg/l, the strain constitutively expressing OmEUG1 and having the antibody gene introduced therein (i.e., the ona09206 strain) exhibited that of 1.4 mg/l, the strain constitutively expressing OmERO1 and having the antibody gene introduced therein (i.e., the ona09406 strain) exhibited that of 2.4 mg/l, the strain constitutively expressing OmKar2 and having the antibody gene introduced therein (i.e., the ona26407 strain) exhibited that of 3.6 mg/l, the strain constitutively expressing OmMPD1 and having the antibody gene introduced therein (i.e., the ona09506 strain) exhibited that of 1.3 mg/l, the strain constitutively expressing OmPDI1 and having the antibody gene introduced therein (i.e., the ona09806 strain) exhibited that of 4.2 mg/l, and the strain constitutively expressing OmHSP104 and having the antibody gene introduced therein (i.e., the ona15807 strain) exhibited that of 1.8 mg/l. By enhancing chaperone expression, the capacity for antibody secretory production was improved by approximately 2 to 5 times. Further, addition of PMT inhibitors to culture was found to improve the amount of antibody secretory production of all the produced strains, as shown in FIG. 7. In the case of the strain constitutively expressing OmPDI1 and having the antibody gene introduced therein (i.e., the ona09806 strain), in particular, the amount of antibody secretory production was found to be improved by approximately 15 times higher than that obtained via control culture of the strain into which the antibody gene had been introduced (i.e., the ona02306 strain) constructed in Example 6. As shown in FIG. 8, it was clearly confirmed via Western blot analysis following non-reducing electrophoresis that a full-length antibody (H2L2) would be secreted and produced.
Example 10
Effects of Chaperone Combination
The expression vector for the anti-TRAIL receptor antibody gene (WO 2001/083560) constructed in Example 2 was introduced into the strain constitutively expressing two types of chaperones bred in Example 5 via electroporation. As an antibody heavy chain, 1 μg of onaP02706 digested with the Sse8387I restriction enzyme was used. As an antibody light chain, 1 μg of onaP03106 digested with the NotI restriction enzyme was used. After electroporation, the transformant was applied on Casamino-U-A agar medium to which ZeocinTM (Invitrogen, R250-01) had been added at a concentration of 100 μg/ml (6.7 g/l of Yeast Nitrogen Base without amino acids, 0.5 g/l of casamino acid, 20 g/l of glucose, 20 mg/l of L-tryptophan, and 20 g/l of Bacto agar), and the transformant was allowed to grow at 30° C. for approximately 2 to 3 days. The grown transformant was allowed to grow again on Casamino-U-A agar medium to which ZeocinTM (Invitrogen, R250-01) had been added at a concentration of 100 μg/ml, and a strain that secretes and produces an antibody was screened for. The ona32507 strain was selected as the strain carrying two OmPDI1 expression units and constitutively expressing OmPDI1 and having the antibody gene introduced therein (i.e., the 2×OmPDI1 strain), the ona33407 strain was selected as the strain constitutively expressing OmPDI1/OmHSP104 and having the antibody gene introduced therein, the ona40007 strain was selected as the strain constitutively expressing OmPDI1/OmKar2 and having the antibody gene introduced therein, and the ona33107 strain was selected as the strain constitutively expressing OmPDI1/OmERO1 and having the antibody gene introduced therein.
As shown in FIG. 9, a control strain into which the antibody gene had been introduced (i.e., the ona02306 strain) exhibited the amount of antibody secretory production of approximately 0.8 mg/l and approximately 1.9 mg/l when PMT inhibitors were added, the strain constitutively expressing OmHSP104 and having the antibody gene introduced therein (i.e., the ona15807 strain) exhibited that of approximately 1.8 mg/l and approximately 4.6 mg/l when PMT inhibitors were added, the strain constitutively expressing OmKar2 and having the antibody gene introduced therein (i.e., the ona26407 strain) exhibited that of approximately 3.6 mg/l and approximately 8.9 mg/l when PMT inhibitors were added, the strain constitutively expressing OmERO1 and having the antibody gene introduced therein (i.e., the ona09406 strain) exhibited that of approximately 2.4 mg/l and approximately 7.6 mg/l when PMT inhibitors were added, and the strain constitutively expressing OmPDI1 and having the antibody gene introduced therein (i.e., the ona09806 strain) exhibited that of approximately 4.2 mg/l and approximately 12.2 mg/l when PMT inhibitors were added. In contrast, the strain constitutively expressing 2×OmPDI1 and having the antibody gene introduced therein (i.e., the ona32507 strain) exhibited the amount of antibody secretory production of approximately 6.8 mg/l and approximately 15.8 mg/l when PMT inhibitors were added, the strain constitutively expressing OmPDI1/OmHSP104 and having the antibody gene introduced therein (i.e., the ona33407 strain) exhibited that of approximately 4.8 mg/l and approximately 13.6 mg/l when PMT inhibitors were added, the strain constitutively expressing OmPDI1/OmKar2 and having the antibody gene introduced therein (i.e., the ona40007 strain) exhibited that of approximately 9.5 mg/l and approximately 18.2 mg/l when PMT inhibitors were added, and the strain constitutively expressing OmPDI1/OmERO1 and having the antibody gene introduced therein (i.e., the ona33107 strain) exhibited that of approximately 17.7 mg/l and approximately 26.2 mg/l when PMT inhibitors were added. The capacity for antibody secretory production that had been improved by approximately 2 to 5 times by enhancing chaperone expression alone was further improved, when chaperone expression was enhanced in combination. When expression of OmPDI1 and that of OmERO1 were enhanced, in particular, the capacity for antibody secretory production was improved by approximately 22 times, and it was improved by approximately 33 times with the addition of PMT inhibitors to culture.
As shown in FIG. 10, it was confirmed via Western blot analysis following non-reducing electrophoresis that the amount of a full-length antibody (H2L2) secreted, which was deduced to be correctly folded, was apparently increased.
Example 11
Effects of Constitutive Expression of Heterologous Protein Disulfide Isomerase (PDI) on Secretory Production of Antibody
The expression vector for the anti-TRAIL receptor antibody gene (WO 2001/083560) constructed in Example 2 was introduced into the strain constitutively expressing heterologous PDI alone bred in Example 5 via electroporation. As an antibody heavy chain, 1 μg of onaP02706 digested with the Sse8387I restriction enzyme was used. As an antibody light chain, 1 μg of onaP03106 digested with the NotI restriction enzyme was used. After electroporation, the transformant was applied on Casamino-U-A agar medium to which ZeocinTM (Invitrogen, R250-01) had been added at a concentration of 100 μg/ml (6.7 g/l of Yeast Nitrogen Base without amino acids, 0.5 g/l of casamino acid, 20 g/l of glucose, 20 mg/l of L-tryptophan, and 20 g/l of Bacto agar), and the transformant was allowed to grow at 30° C. for approximately 2 to 3 days. The grown transformant was allowed to grow again on Casamino-U-A agar medium to which ZeocinTM (Invitrogen, R250-01) had been added at a concentration of 100 μg/ml, and a strain that secretes and produces an antibody was screened for. The ona32207 strain was selected as the strain constitutively expressing Saccharomyces cerevisiae ScPDI1 and having the antibody gene introduced therein, the ona38907 strain was selected as the strain constitutively expressing human PDI and having the antibody gene introduced therein, and the ona39307 strain was selected as the strain constitutively expressing synthetic human PDI (i.e., human PDI with the codon usage being optimized in accordance with that of O. minuta) and having the antibody gene introduced therein.
As shown in FIG. 11, a control strain into which the antibody gene had been introduced (i.e., the ona02306 strain) exhibited the amount of antibody secretory production of approximately 0.8 mg/l and approximately 1.9 mg/l when PMT inhibitors were added, the strain constitutively expressing OmPDI1 and having the antibody gene introduced therein (i.e., the ona09806 strain) exhibited that of approximately 4.2 mg/l and 12.2 mg/l when PMT inhibitors were added, the strain constitutively expressing 2×OmPDI1 and having the antibody gene introduced therein (i.e., the ona32507 strain) exhibited that of approximately 6.8 mg/l and approximately 15.8 mg/l when PMT inhibitors were added, the strain constitutively expressing ScPDI1 and having the antibody gene introduced therein (i.e., the ona32207 strain) exhibited that of approximately 3.7 mg/l and approximately 11.3 mg/l when PMT inhibitors were added, the strain constitutively expressing hPDI and having the antibody gene introduced therein (i.e., the ona38907 strain) exhibited that of approximately 4.0 mg/l and approximately 10.4 mg/l when PMT inhibitors were added, and the strain constitutively expressing synthetic hPDI and having the antibody gene introduced therein (i.e., the ona39307 strain) exhibited that of approximately 9.5 mg/l and approximately 20.1 mg/l when PMT inhibitors were added. When expression of a functional homolog; i.e., heterologous PDI, was enhanced in addition to constitutive expression of O. minuta PDI1, the capacity of antibody secretory production was also found to be improved. In the case of hPDI synthesized by taking the frequency of codon use of O. minuta into consideration, in particular, addition of PMT inhibitors to culture resulted in an improvement in the capacity for antibody secretory production to approximately 25 times higher than that of the control and approximately 1.6 times higher than that obtained upon introduction of OmPDI1 and addition of PMT inhibitors to culture.
Example 12
Effects of Chaperone Combination
The expression vector for the anti-TRAIL receptor antibody gene (WO 2001/083560) constructed in Example 2 was introduced into each of the strain constitutively expressing synthetic human PDI and OmERO1 and the strain constitutively expressing OmPDI1, OmERO1, and OmKar2 bred in Example 5 via electroporation. As an antibody heavy chain, 1 μg of onaP02706 digested with the Sse8387I restriction enzyme was used. As an antibody light chain, 1 μg of onaP03106 digested with the NotI restriction enzyme was used. After electroporation, the transformant was applied on Casamino-U-A agar medium to which ZeocinTM (Invitrogen, R250-01) had been added at a concentration of 100 μg/ml (6.7 g/l of Yeast Nitrogen Base without amino acids, 0.5 g/l of casamino acid, 20 g/l of glucose, 20 mg/l of L-tryptophan, and 20 g/l of Bacto agar), and the transformant was allowed to grow at 30° C. for approximately 2 to 3 days. The grown transformant was allowed to grow again on Casamino-U-A agar medium to which ZeocinTM (Invitrogen, R250-01) had been added at a concentration of 100 μg/ml, and a strain that secretes and produces an antibody was screened for. The ona49707 strain was selected as a strain into which the gene of an antibody constitutively expressing synthetic human PDI/OmERO1 had been introduced, and the ona48707 strain was selected as the strain into which the gene of an antibody constitutively expressing OmPDI1, OmERO1, and OmKar2 had been introduced.
As shown in FIG. 12, a control strain into which the antibody gene had been introduced (i.e., the ona02306 strain) exhibited the amount of antibody secretory production of approximately 0.65 mg/l and approximately 2.2 mg/l when PMT inhibitors were added, the strain constitutively expressing synthetic human PDI and having the antibody gene introduced therein (i.e., the ona39307 strain) exhibited that of approximately 5.1 mg/l and approximately 12.3 mg/l when PMT inhibitors were added, and the strain constitutively expressing OmPDI1/OmERO1 and having the antibody gene introduced therein (i.e., the ona33107 strain) exhibited that of approximately 9.2 mg/l and approximately 18.9 mg/l when f PMT inhibitors were added. In contrast, the strain constitutively expressing synthetic human PDI/OmERO1 and having the antibody gene introduced therein (i.e., the ona49707 strain) exhibited the amount of antibody secretory production of approximately 10.8 mg/l and approximately 23.4 mg/l when PMT inhibitors were added, and the strain constitutively expressing OmPDI1/OmERO1/OmKar2 and having the antibody gene introduced therein (i.e., the ona48707 strain) exhibited that of approximately 16.2 mg/l and approximately 29.8 mg/l when PMT inhibitors were added. When expression of heterologous PDI; i.e., synthetic human PDI and OmERO1, was enhanced, the capacity of antibody secretory production was increased by at least 20%, compared with the case when expression of OmPDI1 and OmERO1 was enhanced. That is, the capacity of antibody secretory production was approximately 36 times higher than that of the control. When expression of OmPDI1, OmERO1, and OmKar2 was enhanced in combination, the capacity of antibody secretory production was further improved, which was approximately 45.8 times higher than that of the control.
Example 13
Construction of Expression Vector for Antibody Gene
In order to express a secretory signal of Kar2 (YJL034W) derived from S. cerevisiae (hereafter referred to as “ScKar2 signal”) and the light and heavy chains of the anti-TRAIL receptor antibody as a fusion protein, the ScKar2 signal gene was ligated to the anti-TRAIL receptor antibody gene (WO 2001/083560) via overlap extension PCR using the oligonucleotide primers below.







For heavy chain of ScKar2 signal-anti-TRAIL


receptor antibody


(SEQ ID NO: 81)


BipXba-F:


5′-GTCTAGATGTTTTTCAACAGACTAAG-3′


 


(SEQ ID NO: 82)


BipTraH-R:


5′-GACTCCACCAGCTGTACTTCAGTTCCGTAGTTTTCTACATC-3′


 


(SEQ ID NO: 83)


BipTraH-F:


5′-GATGTAGAAAACTACGGAACTGAAGTACAGCTGGTGGAGTC-3′


 


(SEQ ID NO: 84)


H04:


5′-GGTCGACTCATTTACCCGGGGACAG-3′


 


For light chain of ScKar2 signal-anti-TRAIL


receptor antibody


(SEQ ID NO: 85)


BipXba-F:


5′-GTCTAGATGTTTTTCAACAGACTAAG-3′


 


(SEQ ID NO: 86)


BipTraL-R:


5′-GATTGGGTCATCTGAATGTCAGTTCCGTAGTTTTCTACATC-3′


 


(SEQ ID NO: 87)


BipTraL-F:


5′-GATGTAGAAAACTACGGAACTGACATTCAGATGACCCAATC-3′


 


(SEQ ID NO: 88)


L04:


5′-GGTCGACCTAACACTCTCCCCTGT-3′






The ScKar2 signal gene region was amplified using genomic DNA of S. cerevisiae prepared using the Y-DER Yeast DNA Extraction Reagent (PIERCE) as a template. PCR was carried out at 95° C. for 10 seconds, 55° C. for 30 seconds, and 68° C. for 60 seconds using the BipXba-F primer and the BipTraH-R primer for the heavy chain, and this cycle was repeated 30 times. Also, PCR was carried out at 95° C. for 10 seconds, 55° C. for 30 seconds, and 68° C. for 60 seconds using the BipXba-F primer and the BipTraL-R primer and AccuPrime Pfx DNA Polymerase (Invitrogen, 12344-024) for the light chain, and this cycle was repeated 30 times. The amplified target DNA fragments of approximately 0.15 kb were recovered.
The antibody gene region was amplified using the anti-TRAIL receptor antibody cDNA (WO 2001/083560) as a template. PCR was carried out at 95° C. for 10 seconds, 55° C. for 30 seconds, and 68° C. for 90 seconds using the BipTraH-F primer and the H04 primer for the heavy chain. Also, PCR was carried out at 95° C. for 10 seconds, 55° C. for 30 seconds, and 68° C. for 90 seconds using the BipTraL-F primer and the L04 primer for the light chain and AccuPrime Pfx DNA Polymerase (Invitrogen, 12344-024), and this cycle was repeated 30 times. The amplified target DNA fragment of a heavy chain region of approximately 1.35 kb and that of a light chain region of approximately 0.65 kb were recovered.
Subsequently, PCR was carried out at 95° C. for 10 seconds, 55° C. for 30 seconds, and 68° C. for 90 seconds using the ScKar2 signal region for the heavy chain and the heavy chain region of approximately 1.35 kb as templates, the BipXba-F primer and the H04 primer, and AccuPrime Pfx DNA Polymerase (Invitrogen, 12344-024), and this cycle was repeated 30 times. The amplified target DNA fragment of approximately 1.5 kb was recovered. Also, PCR was carried out at 95° C. for 10 seconds, 55° C. for 30 seconds, and 68° C. for 60 seconds using the ScKar2 signal region for the light chain and the light chain region of approximately 0.65 kb as templates and the BipXba-F primer and the L04 primer, and this cycle was repeated 30 times. The amplified target DNA fragment of approximately 0.8 kb was recovered. The recovered DNA fragments were cloned into pCR2.1-TOPO. Based on the nucleotide sequence of the inserted DNA fragment, it was confirmed that the target fragments each comprised the gene in which the ScKar signal-antibody heavy chain was fused in frame to the ScKar2 signal-antibody light chain. The XbaI restriction enzyme site introduced into the BipXba-F primer and the SalI restriction enzyme site introduced into the H04 and L04 primers were used to recover a DNA fragment encoding the ScKar2 signal-antibody heavy chain and the ScKar2 signal-antibody light chain via digestion with XbaI and SalI.
In order to express the antibody heavy chain and the antibody light chain in S. cerevisiae, a DNA fragment encoding the ScKar2 signal-antibody heavy chain and the ScKar2 signal-antibody light chain recovered via digestion with XbaI and SalI was ligated to the XbaI-SalI site in the glyceraldehyde-3-phosphoric dehydrogenase gene (TDH3, GAP) promoter-terminator cassette introduced into E. coli/yeast shuttle vector YEp352 (Yeast 2, pp. 163-167, 1986). The resultants were designated as the YEp352GAP-II-ScKarHc and YEp352GAP-II-ScKarLc plasmids.
Subsequently, a gene fragment encoding BamHI-GAP promoter-ScKar2 signal-antibody light chain-GAP terminator-BamHI was recovered from YEp352GAP-II-ScKarLc using the BamHI restriction enzyme sites located at both ends of the GAP promoter-terminator cassette. Subsequently, YEp352GAP-II-ScKarHc was digested with the HpaI restriction enzyme. These fragments were blunt-ended, ligated to each other, and transformed into E. coli JM109. The obtained clones were arbitrarily selected, subjected to colony PCR (94° C. for 30 seconds, 55° C. for 30 seconds, and 72° C. for 60 seconds) using the 352-HpaI-R2 primer (5′-CAAAATGAAGCACAGATGC-3′: SEQ ID NO: 89), the L04 primer (5′-GGTCGACCTAACACTCTCCCCTGT-3′: SEQ ID NO: 88), and TaKaRa LA TaqTM with GC Buffer (Takara Bio, RR02AG), and this cycle was repeated 30 times. Clones into which the GAP promoter-ScKar2 signal-antibody heavy chain-GAP terminator and the GAP promoter-ScKar2 signal-antibody light chain-GAP terminator were inserted in inverse orientation with respect to each other were selected. From the transformants that were confirmed to have been amplified, a vector into which BamHI-GAP promoter-ScKar2 signal-antibody light chain-GAP terminator-BamHI had been introduced at the blunt-ended HpaI restriction enzyme site of YEp352GAP-II-ScKarHc was selected, and it was designated as YEp352 GAP-II-ScKarHc/ScKarLc (FIG. 13).
Example 14
Construction of Expression Vector for Chaperone Gene Alone
(1) Construction of Constitutive Expression Vector for S. cerevisiae PDI1 (Sc PDI1) Gene
PDI1 of Sc (YCL043C: SEQ ID NO: 120 (nucleotide sequence), SEQ ID NO: 121 (amino acid sequence)) was amplified using genomic DNA of S. cerevisiae prepared using the Y-DER Yeast DNA Extraction Reagent (PIERCE) as a template. PCR was carried out using the PDI1-sac primer (5′-CGAGCTCATGAAGTTTTCTGCTGGTG-3′: SEQ ID NO: 90), the PDI1-sma primer (5′-GCCCGGGTTACAATTCATCGTGAATG-3′: SEQ ID NO: 91), and AccuPrime Pfx DNA Polymerase (Invitrogen, 12344-024) at 95° C. for 10 seconds, 55° C. for 30 seconds, and 68° C. for 90 seconds, and this cycle was repeated 30 times. A target DNA fragment of approximately 1.6 kb was recovered and cloned into pCR2.1-TOPO. Thereafter, the nucleotide sequence of the inserted DNA fragment was confirmed. In order to express ScPDI1 in S. cerevisiae, the gene encoding ScPDI1 recovered via digestion with SacI and SmaI with the use of the SacI and SmaI restriction enzyme sites introduced into the PDI1-sac primer and the PDI1-sma primer was ligated to the SacI-SmaI site in the glyceraldehyde-3-phosphoric dehydrogenase gene (TDH3, GAP) promoter-terminator cassette introduced into the E. coli/yeast shuttle vector YEp351 (Yeast 2, pp. 163-167, 1986). The resulting plasmid was designated as YEp351GAP-II-ScPDI1.
(2) Construction of Constitutive Expression Vector for ScMPD1 Gene
ScMPD1 (YOR288C: SEQ ID NO: 122 (nucleotide sequence), SEQ ID NO: 123 (amino acid sequence)) was amplified using genomic DNA of S. cerevisiae prepared using the Y-DER Yeast DNA Extraction Reagent (PIERCE) as a template. PCR was carried out using the MPD1-sac primer (5′-CGAGCTCATGTTATTTCTTAATATTATTAAG-3′: SEQ ID NO: 92), the MPD1-sma primer (5′-GCCCGGGCTACAATTCGTCGTGCTTGTTTCC-3′: SEQ ID NO: 93), and AccuPrime Pfx DNA Polymerase (Invitrogen, 12344-024) at 95° C. for 10 seconds, 55° C. for 30 seconds, and 68° C. for 60 seconds, and this cycle was repeated 30 times. A target DNA fragment of approximately 0.96 kb was recovered and cloned into pCR2.1-TOPO. Thereafter, the nucleotide sequence of the inserted DNA fragment was confirmed. In order to express ScMPD1 in S. cerevisiae, the gene encoding ScMPD1 recovered via digestion with SacI and SmaI with the use of the SacI and SmaI restriction enzyme sites introduced into the MPD1-sac primer and the MPD1-sma primer was ligated to the SacI-SmaI site in the glyceraldehyde-3-phosphoric dehydrogenase gene (TDH3, GAP) promoter-terminator cassette introduced into the E. coli/yeast shuttle vector YEp351 (Yeast 2, pp. 163-167, 1986). The resulting plasmid was designated as YEp351GAP-II-ScMPD1.
(3) Construction of Constitutive Expression Vector for ScSCJ1 Gene
ScSCJ1 (YMR214W: SEQ ID NO: 124 (nucleotide sequence), SEQ ID NO: 125 (amino acid sequence)) was amplified using genomic DNA of S. cerevisiae prepared using the Y-DER Yeast DNA Extraction Reagent (PIERCE) as a template. PCR was carried out using the SCJ1-sac primer (5′-CGAGCTCATGATTCCAAAATTATATATAC-3′: SEQ ID NO: 94), the SCJ1-sma primer (5′-GCCCGGGCTACAACTCATCTTTGAGC-3′: SEQ ID NO: 95), and AccuPrime Pfx DNA Polymerase (Invitrogen, 12344-024) at 95° C. for 10 seconds, 55° C. for 30 seconds, and 68° C. for 60 seconds, and this cycle was repeated 30 times. A target DNA fragment of approximately 1.1 kb was recovered and cloned into pCR2.1-TOPO. Thereafter, the nucleotide sequence of the inserted DNA fragment was confirmed. In order to express ScSCJ1 in S. cerevisiae, the gene encoding ScMPD1 recovered via digestion with SacI and SmaI with the use of the SacI and SmaI restriction enzyme sites introduced into the SCJ1-sac primer and the SCJ1-sma primer was ligated to the SacI-SmaI site in the glyceraldehyde-3-phosphoric dehydrogenase gene (TDH3, GAP) promoter-terminator cassette introduced into the E. coli/yeast shuttle vector YEp351 (Yeast 2, pp. 163-167, 1986). The resulting plasmid was designated as YEp351GAP-II-ScSCJ1.
(4) Construction of Constitutive Expression Vector for ScERO1 Gene
ScERO1 (YML130C: SEQ ID NO: 126 (nucleotide sequence), SEQ ID NO: 127 (amino acid sequence)) was amplified using genomic DNA of S. cerevisiae prepared using the Y-DER Yeast DNA Extraction Reagent (PIERCE) as a template. PCR was carried out using the ERO1-sac2 primer (5′-CGAGCTCATGAGATTAAGAACCGCCATTG-3′: SEQ ID NO: 96), the ERO1-sma2 primer (5′-GCCCGGGTTATTGTATATCTAGCTTATAG-3′: SEQ ID NO: 97), and AccuPrime Pfx DNA Polymerase (Invitrogen, 12344-024) at 95° C. for 10 seconds, 55° C. for 30 seconds, and 68° C. for 120 seconds, and this cycle was repeated 30 times. A target DNA fragment of approximately 1.7 kb was recovered and cloned into pCR2.1-TOPO. Thereafter, the nucleotide sequence of the inserted DNA fragment was confirmed. In order to express ScERO1 in S. cerevisiae, the gene encoding ScERO1 recovered via digestion with SacI and SmaI with the use of the SacI and SmaI restriction enzyme sites introduced into the ERO1-sac2 primer and the ERO1-sma2 primer was ligated to the SacI-SmaI site in the glyceraldehyde-3-phosphoric dehydrogenase gene (TDH3, GAP) promoter-terminator cassette introduced into the E. coli/yeast shuttle vector YEp351 (Yeast 2, pp. 163-167, 1986). The resulting plasmid was designated as YEp351GAP-II-ScERO1.
(5) Construction of Constitutive Expression Vector for ScFKB2 Gene
ScFKB2 (FPR2/YDR519W: SEQ ID NO: 128 (nucleotide sequence), SEQ ID NO: 129 (amino acid sequence)) was amplified using genomic DNA of S. cerevisiae prepared using the Y-DER Yeast DNA Extraction Reagent (PIERCE) as a template. PCR was carried out using the FKB2-sac primer (5′-CGAGCTCATGATGTTTAATATTTACC-3′: SEQ ID NO: 98), the FKB2-sma primer (5′-GCCCGGGCTAGGCGGCTGATTTCACG-3′: SEQ ID NO: 99), and AccuPrime Pfx DNA Polymerase (Invitrogen, 12344-024) at 95° C. for 10 seconds, 55° C. for 30 seconds, and 68° C. for 30 seconds, and this cycle was repeated 30 times. A target DNA fragment of approximately 0.5 kb was recovered and cloned into pCR2.1-TOPO. Thereafter, the nucleotide sequence of the inserted DNA fragment was confirmed. In order to express ScFKB2 in S. cerevisiae, the gene encoding ScFKB2 recovered via digestion with SacI and SmaI with the use of the SacI and SmaI restriction enzyme sites introduced into the FKB2-sac primer and the FKB2-sma primer was ligated to the SacI-SmaI site in the glyceraldehyde-3-phosphoric dehydrogenase gene (TDH3, GAP) promoter-terminator cassette introduced into the E. coli/yeast shuttle vector YEp351 (Yeast 2, pp. 163-167, 1986). The resulting plasmid was designated as YEp351GAP-II-ScFKB2.
(6) Construction of Constitutive Expression Vector for ScJEM1 Gene
ScJEM1 (YJL073W: SEQ ID NO: 130 (nucleotide sequence), SEQ ID NO: 131 (amino acid sequence)) was amplified using genomic DNA of S. cerevisiae prepared using the Y-DER Yeast DNA Extraction Reagent (PIERCE) as a template. PCR was carried out using the JEM1-sac primer (5′-CGAGCTCATGATACTGATCTCGGGATACTGTC-3′: SEQ ID NO: 100), the JEM1-sma primer (5′-GCCCGGGTCAAAGCCCAAAATTCATTTTAAAG-3′: SEQ ID NO: 101), and AccuPrime Pfx DNA Polymerase (Invitrogen, 12344-024) at 95° C. for 10 seconds, 55° C. for 30 seconds, and 68° C. for 120 seconds, and this cycle was repeated 30 times. A target DNA fragment of approximately 1.9 kb was recovered and cloned into pCR2.1-TOPO. Thereafter, the nucleotide sequence of the inserted DNA fragment was confirmed. In order to express ScJEM1 in S. cerevisiae, the gene encoding ScJEM1 recovered via digestion with SacI and SmaI with the use of the SacI and SmaI restriction enzyme sites introduced into the JEM1-sac primer and the JEM1-sma primer was ligated to the SacI-SmaI site in the glyceraldehyde-3-phosphoric dehydrogenase gene (TDH3, GAP) promoter-terminator cassette introduced into the E. coli/yeast shuttle vector YEp351 (Yeast 2, pp. 163-167, 1986). The resulting plasmid was designated as YEp351GAP-II-ScJEM1.
(7) Construction of Constitutive Expression Vector for ScLHS1 Gene
ScLHS1 (YKL073W: SEQ ID NO: 132 (nucleotide sequence), SEQ ID NO: 133 (amino acid sequence)) was amplified using genomic DNA of S. cerevisiae prepared using the Y-DER Yeast DNA Extraction Reagent (PIERCE) as a template. PCR was carried out using the LHS1.1-sac primer (5′-CGAGCTCATGCGAAACGTTTTAAGGCTTT-3′: SEQ ID NO: 102), the LHS1-sma primer (5′-GCCCGGGCTATAATTCATCATGCAAAATG-3′: SEQ ID NO: 103), and AccuPrime Pfx DNA Polymerase (Invitrogen, 12344-024) at 95° C. for 10 seconds, 55° C. for 30 seconds, and 68° C. for 180 seconds, and this cycle was repeated 30 times. A target DNA fragment of approximately 2.65 kb was recovered and cloned into pCR2.1-TOPO. Thereafter, the nucleotide sequence of the inserted DNA fragment was confirmed. In order to express ScLHS1 in S. cerevisiae, the gene encoding ScLHS1 recovered via digestion with SacI and SmaI with the use of the SacI and SmaI restriction enzyme sites introduced into the LHS1.1-sac primer and the LHS1-sma primer was ligated to the SacI-SmaI site in the glyceraldehyde-3-phosphoric dehydrogenase gene (TDH3, GAP) promoter-terminator cassette introduced into the E. coli/yeast shuttle vector YEp351 (Yeast 2, pp. 163-167, 1986). The resulting plasmid was designated as YEp351GAP-II-ScLHS1.
(8) Construction of Constitutive Expression Vector for ScMPD2 Gene
ScMPD2 (YOL088C: SEQ ID NO: 134 (nucleotide sequence), SEQ ID NO: 135 (amino acid sequence)) was amplified using genomic DNA of S. cerevisiae prepared using the Y-DER Yeast DNA Extraction Reagent (PIERCE) as a template. PCR was carried out using the MPD2-sac primer (5′-CGAGCTCATGAAATTGCACGGCTTTTTATTTTC-3′: SEQ ID NO: 104), the MPD2-sma primer (5′-GCCCGGGTCAAAGCTCGTCATGACTACTGG-3′: SEQ ID NO: 105), and AccuPrime Pfx DNA Polymerase (Invitrogen, 12344-024) at 95° C. for 10 seconds, 55° C. for 30 seconds, and 68° C. for 60 seconds, and this cycle was repeated 30 times. A target DNA fragment of approximately 0.8 kb was recovered and cloned into pCR2.1-TOPO. Thereafter, the nucleotide sequence of the inserted DNA fragment was confirmed. In order to express Sc MPD2 in S. cerevisiae, the gene encoding ScMPD2 recovered via digestion with SacI and SmaI with the use of the SacI and SmaI restriction enzyme sites introduced into the MPD2-sac primer and the MPD2-sma primer was ligated to the SacI-SmaI site in the glyceraldehyde-3-phosphoric dehydrogenase gene (TDH3, GAP) promoter-terminator cassette introduced into the E. coli/yeast shuttle vector YEp351 (Yeast 2, pp. 163-167, 1986). The resulting plasmid was designated as YEp351 GAP-II-ScMPD2.
(9) Construction of Constitutive Expression Vector for ScERJ5 Gene
ScERJ5 (YFR041C: SEQ ID NO: 136 (nucleotide sequence), SEQ ID NO: 137 (amino acid sequence)) was amplified using genomic DNA of S. cerevisiae prepared using the Y-DER Yeast DNA Extraction Reagent (PIERCE) as a template. PCR was carried out using the YFR041C-sac primer (5′-CGAGCTCATGAACGGTTACTGGAAAC-3′: SEQ ID NO: 106), the YFR041C-sma primer (5′-GCCCGGGTCATTTACGTGAATAGATC-3′: SEQ ID NO: 107), and AccuPrime Pfx DNA Polymerase (Invitrogen, 12344-024) at 95° C. for 10 seconds, 55° C. for 30 seconds, and 68° C. for 60 seconds, and this cycle was repeated 30 times. A target DNA fragment of approximately 0.9 kb was recovered and cloned into pCR2.1-TOPO. Thereafter, the nucleotide sequence of the inserted DNA fragment was confirmed. In order to express ScERJ5 in S. cerevisiae, the gene encoding ScERJ5 recovered via digestion with SacI and SmaI with the use of the SacI and SmaI restriction enzyme sites introduced into the YFR041C-sac primer and the YFR041C-sma primer was ligated to the SacI-SmaI site in the glyceraldehyde-3-phosphoric dehydrogenase gene (TDH3, GAP) promoter-terminator cassette introduced into the E. coli/yeast shuttle vector YEp351 (Yeast 2, pp. 163-167, 1986). The resulting plasmid was designated as YEp351GAP-II-ScERJ5.
(10) Construction of Constitutive Expression Vector for ScEUG1 Gene
ScEUG1 (YDR518W: SEQ ID NO: 138 (nucleotide sequence), SEQ ID NO: 139 (amino acid sequence)) was amplified using genomic DNA of S. cerevisiae prepared using the Y-DER Yeast DNA Extraction Reagent (PIERCE) as a template. PCR was carried out using the EUG1-sac primer (5′-CGAGCTCATGCAAGTGACCACAAGATT-3′: SEQ ID NO: 108), the EUG1-sma primer (5′-GCCCGGGTTATAATTCATCATGTACGG-3′: SEQ ID NO: 109), and AccuPrime Pfx DNA Polymerase (Invitrogen, 12344-024) at 95° C. for 10 seconds, 55° C. for 30 seconds, and 68° C. for 90 seconds, and this cycle was repeated 30 times. A target DNA fragment of approximately 1.5 kb was recovered and cloned into pCR2.1-TOPO. Thereafter, the nucleotide sequence of the inserted DNA fragment was confirmed. In order to express ScEUG1 in S. cerevisiae, the gene encoding ScEUG1 recovered via digestion with SacI and SmaI with the use of the SacI and SmaI restriction enzyme sites introduced into the EUG1-sac primer and the EUG1-sma primer was ligated to the SacI-SmaI site in the glyceraldehyde-3-phosphoric dehydrogenase gene (TDH3, GAP) promoter-terminator cassette introduced into the E. coli/yeast shuttle vector YEp351 (Yeast 2, pp. 163-167, 1986). The resulting plasmid was designated as YEp351GAP-II-ScEUG1.
Example 15
Effects of Chaperone on Antibody Secretory Production
Competent cells of the ZUO1/YGR285C strain (MATa Δhis3 Δleu2 Δmet15 Δura3Δ zuo1) derived from the S. cerevisiae BY4741 strain were prepared using the Frozen-EZ Yeast Transformation II Kit (ZYMO RESARCH). The S. cerevisiae ZUO1/YGR285C strain was sowed in 5 ml of YPAD medium (YPD medium containing 0.04% adenine (Sigma)), and yeast cells resulting from overnight culture at 30° C. and 310 rpm were used. The expression vectors constructed in Example 13 and in Example 14 were introduced into the S. cerevisiae ZUO1/YGR285C strain using the Frozen-EZ Yeast Transformation II Kit (ZYMO RESARCH), and transformants grown on ST agar medium containing 2% agar (the Yeast Nitrogen Base and Ammonium sulfate medium (Sigma) containing 2% glucose, 0.04% adenine, and 0.3M KCl and lacking uracil and leucine) were each selected as a yeast strain expressing an antibody constitutively expressing chaperone.
YEp352 GAP-II-ScKarHc/ScKarLc carrying the antibody heavy chain and light chain expression unit comprises an URA3 marker gene that complements uracil-requiring mutation of a host. In contrast, YEp351 GAP-II (a control vector into which the gene is not introduced), YEp351GAP-II-ScPDI1 (the ScPDI1 expression vector), YEp351GAP-II-ScMPD1 (the ScMPD1 expression vector), YEp351GAP-II-ScSCJ1 (the ScSCJ1 expression vector), YEp351GAP-II-ScERO1 (the ScERO1 expression vector), YEp351GAP-II-ScFKB2 (the ScFKB2 expression vector), YEp351GAP-II-ScJEM1 (the ScJEM1 expression vector), YEp351GAP-II-ScLHS1 (the ScLHS1 expression vector), YEp351GAP-II-ScMPD2 (ScMPD2 expression vector), YEp351GAP-II-ScERJ5 (the ScERJ5 expression vector), and YEp351GAP-II-ScEUG1 (the ScEUG1 expression vector) each has a LEU2 marker gene that complements leucine-requiring mutation of a host. Host cells were transformed so that they can grow only when the expression vector for the gene of an antibody and the constitutive expression vector for chaperone are introduced, and 10 types of yeast strains expressing an antibody constitutively expressing chaperone were constructed from the S. cerevisiae ZUO1/YGR285C strain.
ST medium (500 μl) (Yeast Nitrogen Base and Ammonium sulfate medium containing 2% glucose, 0.04% adenine, and 0.3M KCl and lacking uracil and leucine (Sigma)) was applied onto a 96-deep well plate (Greiner, 780271), the transformant was sowed with the use of a toothpick, the top of the plate was sealed with CO2-permeable plate sealers (Greiner, 676051), and culture was carried out at an agitation speed of 310 rpm, an amplitude of 50 mm, at 30° C. for 3 days. Control culture was carried out by applying 1 ml of YPAD medium to a 96-deep well plate (Greiner, 780271). In the case of culture involving the addition of PMT inhibitors, 1 ml of YPAD medium to which the rhodanine-3-acetic acid derivative 1c had been initially added to a final concentration of 10 μM was applied to the plate. The culture solution that had been cultured in ST medium was sowed to a final concentration of 5%, and the top of the plate was sealed with CO2-permeable plate sealers (Greiner, 676051). Thereafter, culture was carried out at an agitation speed of 310 rpm, an amplitude of 50 mm, at 30° C. for 3 days. The culture supernatant was prepared from the culture solution and it was designated as a sample containing an antibody secreted and produced by yeast. Antibody secretory production was confirmed via sandwich ELISA. Yeast cells were removed from the culture product via centrifugation at 2,700×g and 4° C. for 5 minutes, and the resulting culture supernatant was designated as a sample of secreted antibody. Sandwich ELISA was carried out by adsorbing the TRAIL receptor proteins that were antigens of the anti-TRAIL receptor antibodies on a 96-well plate, adding the sample of secreted antibody, and performing detection using a peroxidase-labeled human IgG specific Fc antibody (Peroxidase-labeled affinity purified antibody to human IgG (Fc) (KPL, 04-10-20)) and the ABTS peroxidase substrate (KPL, 50-66-01).
As shown in FIG. 14, a control strain exhibited the amount of antibody secretory production of 3.9 ng/ml. When addition of an O-sugar chain was suppressed, however, the control strain exhibited an approximately 2.5 times greater amount of antibody secretory production; i.e., 10 ng/ml, and effects of suppression of O-sugar chain addition were observed. When addition of an O-sugar chain was suppressed, the strains into which each of ScERO1 and ScPDI1 had been introduced each exhibited approximately 7 times and approximately 20 times greater amounts of antibody secretory production; i.e., 26.4 ng/ml and 79.2 ng/ml.
Example 16
Construction of Coexpression Strain for OmPDI1 and Human Alpha-2,3-Sialic Acid Transferase (ST3GalI)
(1) Construction of ST3GalI Expression Vector
A gene encoding a sequence comprising 3 repeats of the FLAG sequence (3× FLAG) was amplified. The gene was amplified via PCR using the P3× FLAG-CMV-13 expression vector (Sigma, E4776) as a template and the 5′-3×FLAG primer (5′-atcatcatcatgcccgggccgactacaaagaccat-3′: SEQ ID NO: 110), the 3′-3× FLAG primer (5′-gcaccgtctcggatcccttgtcatcgtcatcctt-3′: SEQ ID NO: 111) to which the SrfI site and the BamHI site had been added, respectively. The resulting fragment was inserted into the O. minuta expression vector, pOMEA1-His6, (Akeboshi et al., 2007, Appl. Environ. Microbiol., 73:4805-4812) cleaved with SrfI and BamHI using the In-Fusion Dry-Down PCR Cloning Kit (Takara Bio, Z9602N). The resulting vector was designated as pOMEA1-6H3F.
Subsequently, a gene encoding a catalyst site of ST3GalI was amplified. The ST3GalI gene is present in human chromosome 8 and its cDNA nucleotide sequence is registered with a public database, GenBank, under the Accession No. L29555. The catalyst domain of the ST3GalI gene was first amplified via PCR using the 3FLAG-ST3Gal1-F primer (5′-GATGACGATGACAAGGGATCCaactactcccacaccatgg-3′: SEQ ID NO: 112) and the 3FLAG-ST3Gal1-R primer (5′-GCACCGTCTCGGATCCtcatctccccttgaagatccggat-3′: SEQ ID NO: 113). An entry clone of human glycosyltransferase library prepared in the NEDO SG project was used as a template (Shimma et al., 2006, Appl. Environ. Microbiol., 72, 7003-7012). The obtained fragment was inserted into the BamHI site of pOMEA1-6H3F using the In-Fusion Dry-Down PCR Cloning Kit (Takara Bio, Z9602N). After the nucleotide sequence was confirmed, the fragment was designated as the expression vector, pOMEA1-6H3F-ST3Gal1.
(2) Construction of ST3GalI Expression Strain
The pOMEA1-6H3F-ST3Gal1 plasmid was cleaved with the NotI restriction enzyme, and the O. minuta TK10-1-2 strain was then transformed. Transformation was carried out by the lithium acetate method. After transformation, the resultant was applied on SD-Ade medium (2% glucose, 0.17% Yeast Nitrogen Base without amino acids (Difco), nucleic acid bases except for adenine, and amino acids mixture (20 to 400 mg/l)), and culture was carried out at 30° C. for 2 days. Whether or not the expression vector was incorporated into a chromosome was confirmed via colony PCR described in Example 5 and the transformant was designated as the YT-1 strain.
Subsequently, the O. minuta-derived PDI1 expression vector (onaP03606) constructed in Example 3 was introduced. Also, transformation was carried out using pOMexGP1U containing no OmPDI1 as a control. After the YT-1 strain was transformed in the manner described above, the transformant was applied on SD-Ura medium (2% glucose, 0.17% Yeast Nitrogen Base without amino acids (Difco), nucleic acid bases except for uracil, and amino acid mixture (20 to 400 mg/l)), and culture was carried out at 30° C. for 2 days. Whether or not the expression vector was incorporated into a chromosome was confirmed via colony PCR described in Example 5, the strain into which pOMexGP1U (control plasmid) had been introduced was designated as the YT-2 strain, and the strain into which onaP03606 had been introduced was designated as the YT-3 strain.
Example 17
Expression of Human Alpha-2,3-Sialic Acid Transferase (ST3GalI)
The YT-2 strain and the YT-3 strain were inoculated in 5 ml of YPAD+KCl medium (2% polypeptone, 1% yeast extract, 2% glucose, adenine (40 mg/l), 0.3 M potassium chloride), and preculture was carried out at 30° C. overnight. Subsequently, 1 ml of a preculture solution was inoculated in 150 ml of YPAD+KCl medium, and culture was carried out at 30° C. for 48 hours. The strains were recovered, resuspended in 100 ml of BMMY+2% casamino acid medium (1% yeast extract, 2% polypeptone, 1.34% Yeast Nitrogen Base without amino acids (Difco), 0.1 M KPi (pH 6.0), 2% casamino acid, 1% methanol), and cultured at 20° C. for 96 hours. Methanol was added to a concentration of 0.5% every 12 hours. After the completion of culture, the culture product was subjected to centrifugation to remove yeast cells, and the resulting solution was designated as a crude enzyme solution.
Enzyme activity was assayed in the following manner. A crude enzyme solution (2 μl) was added to 18 μl of a reaction solution (0.1 M Tris-HCl (pH 7.5), 1 mM MnCl2, 5 mM CMP-NeuAc, 50 μM core 1-pNP), and the reaction was initiated. After the reaction was carried out at 37° C. for 30 minutes, the reaction was terminated via boiling, and HPLC analysis was carried out. A Cosmosil 5C18-ARII column (4.6×250 mm, Nacalai Tesque, Inc.) was used, and a mixture of 0.2 M triethylamine-acetic acid (pH 7.0, solution A) and acetonitrile (solution B) (solution A:solution B=9:1) was used as a mobile phase. The column was equilibrated, the sample was injected therein, and elution was carried out 20 minutes thereafter. Detection was carried out using a UV detector (detection wavelength: 300 nm). A core1-pNP substrate was eluted 14.5 minutes later, and a reaction product, sialylcore1-pNP, was eluted 17 minutes later. The reaction product was quantified based on the peak area, and the result was designated as activity (U). The unit “1 U” was defined as the amount of an enzyme that is necessary to produce 1 μmol of the reaction product per minute. FIG. 15 shows changes in expression levels of the enzyme with the elapse of time during culture. As a result, increased enzyme activity was observed in the ST3GalI-expressing strain (YT-3) into which OmPDI1 had been introduced, compared with the strain into which no OmPDI1 had been introduced (YT-2), and difference in the expression levels became as great as approximately 3.4 times 96 hours later.
Western blot analysis was then carried out. The culture supernatants in amounts equivalent to “10 μU” of the YT-2 strain and the YT-3 strain were subjected to treatment with EndoHf (NEB, P0703S) in accordance with the protocol, applied, and then subjected to SDS-PAGE. After electrophoresis, the product was transferred to a PVDF membrane, and Western blotting was carried out in accordance with a conventional technique. A mouse anti-FLAG M2 antibody (Sigma, F1804) was used as a primary antibody and an anti-mouse IgG antibody-alkaline phosphatase composite was used as a secondary antibody. Detection was carried out using CDP-Star and an image analyzer, LAS1000 (Fujifilm Corporation). Further, signals were quantified using Image Gauge (Fujifilm Corporation).
The results are shown in FIG. 16 and in Table 1. Even though Western blot analysis was carried out in addition to enzyme activity assay, the intensity of the ST3GalI signal derived from the strain into which OmPDI1 had been introduced (YT-3) was found to be attenuated to as low as 0.6 times. It was thus considered that introduction of OmPDI1 resulted in promotion of normal disulfide bond formation in ST3GalI molecules, and the number of correctly folded proteins was increased, which in turn elevated the specific activity by approximately 1.6 times.











TABLE 1







Chaperone-expressing
Signal intensity
Specific activity



strain
(AU)
(pU/AU)









YT-2
4694842
2.13



YT-3
2976349
3.36










Example 18
Construction of Coexpression Strain for an Expression Vector for Chaperone Gene Combination and an Expression Vector for Human Alpha-2,3-Sialic Acid Transferase (ST3GalI)
The expression vector for various chaperone gene combinations derived from O. minuta prepared in Example 4 was introduced into the YT-1 strain of Example 17. In the same manner as in Example 16, plasmids were cleaved with NotI to transform the YT-1 strain. After transformation, the resultant was applied on SD-Ura medium (2% glucose, 0.17% Yeast Nitrogen Base without amino acids (Difco), nucleic acid bases except for uracil, and amino acid mixture (20 to 400 mg/l)) and culture was carried out at 30° C. for 2 days. Whether or not the expression vector was incorporated into a chromosome was confirmed via colony PCR described in Example 5, an expressed strain having a coexpression vector (onaP09407) carrying two copies of the OmPDI1 expression cassette was designated as the YT-4 strain, an expressed strain having a coexpression vector (onaP09707) for OmPDI1 and OmKar2 was designated as the YT-5 strain, an expressed strain having a coexpression vector (onaP09507) for OmPDI1 and OmERO1 was designated as the YT-6 strain, and an expressed strain having a coexpression vector (onaP09607) for OmPDI1 and OmHSP104 was designated as the YT-7 strain. Culture was carried out in the same manner as in Example 17, enzyme expression from each strain was assayed with the elapse of time, and the results are shown in FIG. 17. Also, 30 μl of the culture supernatant was subjected to treatment with EndoHf (NEB, P0703S) in accordance with the protocol and then Western blot analysis. The results are shown in FIG. 18, and quantified signal intensities and specific activities determined based thereon are shown in Table 2.










TABLE 2





Chaperone-expressing
Signal intensity
Activity
Specific activity


strain
(AU)
(mU/ml)
(pU/AU)


















YT-4-1
5003576.86
2.32
13.9


YT-4-2
5967631.86
4.50
23.7


YT-4-3
6004529.86
4.47
22.3


YT-4-4
5686573.24
5.08
26.8


YT-5-1
3619105.78
1.29
10.7


YT-5-2
3926906.78
2.07
15.8


YT-5-3
4530942.78
1.86
12.3


YT-5-4
4603909.78
2.29
14.9


YT-6-1
2225292.78
0.70
9.4


YT-6-2
4312501.78
3.10
21.6


YT-6-3
4786891.78
5.03
31.5


YT-6-4
4057802.78
6.81
50.3


YT-7-1
3381700.78
1.28
11.4


YT-7-2
3208548.78
2.98
27.9


YT-7-3
2435370.00
3.45
42.5


YT-7-4
1912043.00
3.65
57.3









Enzyme expression levels substantially stopped increasing 2 days after induction in the YT-4, -5, and -7 strains; however, activity kept increasing at significant levels up to 4 days after induction in the YT-6 strain, and activity per ml of the culture supernatant of the YT-6 strain was the highest at the end. Although no clear increase in specific activity was observed in the YT-4 and YT-5 expression strains, an increase was observed in specific activity with the elapse of time in the YT-6 and YT -7 strains. Thus, it was considered that coexpression of ERO1 or HSP104 would result in an increase in the number of enzyme molecules that were correctly folded.
Example 19
Construction of Expression Vector for Chaperone Gene Alone
(1) Construction of Constitutive Expression Vector for Chinese Hamster GRP78/BiP Gene
The Chinese hamster GRP78 gene (Accession No. M17169: SEQ ID NO: 152) was cloned in the following manner to construct an expression vector.
At the outset, total RNA was extracted from the CHO/dhFr-strain (ATCC CRL-9096) cell obtained via culture in Ex325 medium with the use of the QuickPrep total RNA Extraction Kit (GE Healthcare Life Sciences, 27-9271-01). Purified total RNA (600 ng) was used to synthesize cDNA via reverse transcription using ReverTra Ace-α-(TOYOBO, FSK-101). PCR was carried out using the resulting cDNA as a template, the CHOGRP78-F primer (5′-ACCCTCGAGATGAAGTTCCCTATGGTGG-3′: SEQ ID NO: 114), the CHOGRP78-R primer (5′-ACCGCGGCCGCCTACAACTCATC-3′: SEQ ID NO: 115), and AccuPrime Pfx DNA Polymerase (Invitrogen, 12344-024) at 95° C. for 10 seconds, 55° C. for 30 seconds, and 68° C. for 120 seconds, and this cycle was repeated 30 times. A target DNA fragment of approximately 2 kb was recovered and cloned into pCR2.1-TOPO. Thereafter, the nucleotide sequence of the inserted DNA fragment was confirmed. The CHO GRP78 gene recovered via digestion with XhoI and NotI was ligated to the XhoI-NotI site in pME18s (Accession No. AB009864) with the use of the XhoI and NotI restriction enzyme sites introduced into the CHOGRP78-F primer and the CHOGRP78-R primer. The resulting plasmid was designated as pME18s/choGRP78.
(2) Construction of Constitutive Expression Vector for Chinese Hamster PDI Gene
The Chinese hamster PDI gene (Accession No. AF364317: SEQ ID NO: 153) was cloned in the following manner to construct an expression vector.
PCR was carried out using the resulting cDNA as a template as in the case of (1) above, the CHOPDI-F primer (5′-ACCCTCGAGATGCTGAGCCGTTCTC-3′: SEQ ID NO: 116), the CHOPDI-R primer (5′-ACCGCGGCCGCCTACAATTCGTCCTTTAC-3′: SEQ ID NO: 117), and AccuPrime Pfx DNA Polymerase (Invitrogen, 12344-024) at 95° C. for 10 seconds, 55° C. for 30 seconds, and 68° C. for 90 seconds, and this cycle was repeated 30 times. A target DNA fragment of approximately 1.5 kb was recovered and cloned into pCR2.1-TOPO. Thereafter, the nucleotide sequence of the inserted DNA fragment was confirmed. The CHO PDI gene recovered via digestion with XhoI and NotI was ligated to the XhoI-NotI site in pME18s (Accession No. AB009864) with the use of the XhoI and NotI restriction enzyme sites introduced into the CHOPDI-F primer and the CHOPDI-R primer. The resulting plasmid was designated as pME18s/choPDI.
(3) Construction of Constitutive Expression Vector for Human ERO1-Lβ Gene
The human ERO1-Lβ gene (Accession No. NM—019891: SEQ ID NO: 146) was cloned in the following manner to construct an expression vector.
At the outset, total RNA was extracted from the FreeStyle 293-F cell (Invitrogen, R790-07) obtained via culture in Freestyle 293 Medium (Invitrogen, 12338-018) with the use of the QuickPrep total RNA Extraction Kit (GE Healthcare Life Sciences, 27-9271-01). Purified total RNA (600 ng) was used to synthesize cDNA via reverse transcription using ReverTra Ace-α-(TOYOBO, FSK-101). PCR was carried out using the resulting cDNA as a template, the hERO-F primer (5′-ACCCTCGAGATGAGCCAAGGG-3′: SEQ ID NO: 118), the hERO-R primer (5′-ACCCTCGAGTTACCTACTGTGTTGTAATAAGAC-3′: SEQ ID NO: 119), and AccuPrime Pfx DNA Polymerase (Invitrogen, 12344-024) at 95° C. for 10 seconds, 55° C. for 30 seconds, and 68° C. for 90 seconds, and this cycle was repeated 30 times. A target DNA fragment of approximately 1.4 kb was recovered and cloned into pCR2.1-TOPO. Thereafter, the nucleotide sequence of the inserted DNA fragment was confirmed. The human ERO1-Lβ gene recovered via digestion with XhoI was ligated to the XhoI site of pME18s (Accession No. AB009864) with the use of the XhoI restriction enzyme site introduced into the hERO-F primer and the hERO-R primer. The resulting plasmid was designated as pME18s/hERO1-Lβ.
(4) Construction of Antibody Expression Vector
The light chain and the heavy chain of the anti-TRAIL receptor antibody gene (WO 2001/083560) was introduced into the pcDNA3.1 (+) expression vector (Invitrogen, V790-20), and the resultant was designated as pcDNA3.1 (+)/expAB.
Example 20
Effects of Chaperone on Antibody Secretory Production
The constitutive expression vectors for various chaperone genes constructed in Example 19 (1) to (3) were introduced into the COS-1 cells to examine the effects of chaperone introduction on secretory production of antibody.
The COS-1 cells were sowed on a 24-well microplate (IWAKI SI-Tech, 3820-024N) at 1.5×105 cells/well, and the anti-TRAIL receptor antibody-expressing vector, pcDNA3.1 (+)/expAB, and the constitutive expression vectors for various chaperone genes were cotransfected with the use of Lipofectamine 2000 (Invitrogen, 11668-027) 24 hours after the initiation of culture. After the gene was introduced, culture was carried out for 48 hours, the culture supernatant was clarified via centrifugation at 21,600×g at 4° C. for 3 minutes, and the resultant was designated as a secretory antibody sample.
The secreted and produced antibodies were subjected to quantitative assay via sandwich ELISA. The TRAIL receptor protein (i.e., the antigen of the anti-TRAIL receptor antibody) was adsorbed on a 96-well plate, the sample of secreted antibody 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, 04-10-20)) and the ABTS peroxidase substrate (KPL, 50-66-01). As shown in FIG. 19, improvement in the amount of production by approximately 19% and approximately 13% was observed in the CHO BiP-introduced strain and the CHO PDI-introduced strain, compared with the control strain (i.e., a strain into which only the antibody gene was introduced). When chaperone genes were introduced in combination, also, the amount of secretory production was found to increase by approximately 30% in the strain into which CHO BiP+human ERO1-Lβ and CHO PDI+human ERO1-Lβ had been introduced, compared with the control strain (i.e., a strain into which only the antibody gene was introduced).
Example 21
Construction of Expression Vector for Chaperone Gene Alone
(1) Construction of Constitutive Expression Vector for Human ERO1-Lα gene
Human ERO1-Lα (hERO1-Lα, Accession No. Q96HE7) is a functional homolog of OmERO1 and it comprises an amino acid sequence composed of 468 amino acid residues (SEQ ID NO: 144) encoded by a 1,407-bp nucleotide sequence (SEQ ID NO: 143). The hERO1-Lα gene was synthesized by taking codon preference of O. minuta into consideration (Operon Biotechnologies). From a plasmid carrying the DNA fragment with a confirmed nucleotide sequence, a DNA fragment (SEQ ID NO: 145) containing synthetic hERO1-Lα was recovered via digestion with SalI and EcoT22I with the use of the SalI restriction enzyme site and the EcoT22I restriction enzyme site that had been introduced at the time of synthesis. In order to constitutively express synthetic hERO1-Lα in O. minuta, hERO1-Lα was ligated to pOMexGP1U recovered via digestion with SalI and EcoT22I disclosed in WO 2003/091431. The resulting plasmid was designated as onaP17608.
(2) Construction of Constitutive Expression Vector for Human ERO1-Lβ Gene
Human ERO1-Lβ (hERO1-Lβ, Accession No. NP—063944) is a functional homolog of OmERO1 and it comprises an amino acid sequence composed of 467 amino acid residues (SEQ ID NO: 147) encoded by a 1,404-bp nucleotide sequence (SEQ ID NO: 146). The hERO1-Lβ gene was synthesized by taking codon preference of O. minuta into consideration (Operon Biotechnologies). From a plasmid carrying the DNA fragment with a confirmed nucleotide sequence, a DNA fragment (SEQ ID NO: 148) containing synthetic hERO1-Lα was recovered via digestion with SalI and EcoT22I with the use of the SalI restriction enzyme site and the EcoT22I restriction enzyme site that had been introduced at the time of synthesis. In order to constitutively express synthetic hERO1-Lβ in O. minuta, synthetic hERO1-Lβ was ligated to pOMexGP1U recovered via digestion with SalI and EcoT22I disclosed in WO 2003/091431. The resulting plasmid was designated as onaP17808.
(3) Construction of Constitutive Expression Vector for Human GRP78 Gene
Human GRP78 (hGRP78, Accession No. NP—005338) is a functional homolog of OmKar2 and it comprises an amino acid sequence composed of 654 amino acid residues (SEQ ID NO: 150) encoded by a 1,965-bp nucleotide sequence (SEQ ID NO: 149). The hGRP78 gene was synthesized by taking codon preference of O. minuta into consideration (Operon Biotechnologies). From a plasmid carrying the DNA fragment with a confirmed nucleotide sequence, a DNA fragment (SEQ ID NO: 151) containing synthetic hGRP78 was recovered via digestion with SalI and EcoT22I with the use of the SalI restriction enzyme site and the EcoT22I restriction enzyme site that had been introduced at the time of synthesis. In order to constitutively express synthetic hGRP78 in O. minuta, synthetic hGRP78 was ligated to pOMexGP1U recovered via digestion with SalI and EcoT22I disclosed in WO 2003/091431. The resulting plasmid was designated as onaP17408.
Example 22
Construction of Expression Vector for Chaperone Gene Combination
(1) Construction of Coexpression and Constitutive Expression Vector for Synthetic hPDI Gene and Synthetic hERO1-Lα Gene
The synthetic hERO1-Lα region was recovered from the constitutive expression vector for synthetic hERO1-Lα, onaP17608, with the use of SalI and EcoT22I restriction enzymes. After the pZ/GpGt vector constructed in Example 4 was digested with the SalI and EcoT22I restriction enzymes, the SalI-EcoT22I fragment containing synthetic hERO1-Lα was introduced to construct pZ/GpGt/synthetic hERO1-Lα. Subsequently, pZ/GpGt/synthetic hERO1-Lα was digested with the ApaI restriction enzyme, and a fragment containing GAP promoter-synthetic hERO1-Lα-GAP terminator (the synthetic hERO1-Lα expression cassette) was recovered. The recovered synthetic hERO1-Lα expression cassette was introduced into the ApaI restriction enzyme of the synthetic hPDI expression vector (onaP09107), the insertion direction was confirmed via PCR, and a vector into which the synthetic hPDI expression cassette and synthetic hERO1-Lα expression cassette was introduced in inverse orientation with respect to each other around the URA3 marker of the synthetic hPDI expression vector (onaP09107) was selected. The resulting coexpression vector for synthetic hPDI and synthetic hERO1-Lα was designated as onaP18208.
(2) Construction of Coexpression and Constitutive Expression Vector for Synthetic hPDI Gene and Synthetic hERO1-Lβ Gene
The synthetic hERO1-Lβ region was recovered from the constitutive expression vector for synthetic hERO1-Lβ, onaP17608, with the use of SalI and EcoT22I restriction enzymes. After the pZ/GpGt vector constructed in Example 4 was digested with the SalI and EcoT22I restriction enzymes, the SalI-EcoT22I fragment containing synthetic hERO1-Lβ was introduced to construct pZ/GpGt/synthetic hERO1-Lβ. Subsequently, pZ/GpGt/synthetic hERO1-Lβ was digested with the ApaI restriction enzyme, and a fragment containing the GAP promoter-synthetic hERO1-Lβ-GAP terminator (the synthetic hERO1-Lβ expression cassette) was recovered. The recovered synthetic hERO1-Lβ expression cassette was introduced into the ApaI restriction enzyme of the synthetic hPDI expression vector (onaP09107), the insertion direction was confirmed via PCR, and a vector into which the synthetic hPDI expression cassette and the synthetic hERO1-Lβ expression cassette were inserted in inverse orientation with respect to each other around the URA3 marker of the synthetic hPDI expression vector (onaP09107) was selected. The resulting coexpression vector for synthetic hPDI1 and synthetic hERO1-Lβ was designated as onaP18308.
(3) Construction of Coexpression and Constitutive Expression Vector for Synthetic hPDI Gene and Synthetic hGRP78 Gene
The synthetic hGRP78 region was recovered from the constitutive expression vector for synthetic hGRP78, onaP17408, with the use of SalI and EcoT22I restriction enzymes. After the pZ/GpGt vector constructed in Example 4 was digested with the SalI and EcoT22I restriction enzymes, the SalI-EcoT22I fragment containing synthetic hGRP78 was introduced to construct pZ/GpGt/synthetic hGRP78. Subsequently, pZ/GpGt/synthetic hGRP78 was digested with the ApaI restriction enzyme, and a fragment containing the GAP promoter-synthetic hGRP78-GAP terminator (the synthetic hGRP78 expression cassette) was recovered. The recovered synthetic hGRP78 expression cassette was introduced into the ApaI restriction enzyme of the synthetic hPDI expression vector (onaP09107), the insertion direction was confirmed via PCR, and a vector into which the synthetic hPDI expression cassette and synthetic hGRP78 expression cassette were inserted in inverse orientation with respect to each other around the URA3 marker of the synthetic hPDI expression vector (onaP09107) was selected. The resulting coexpression vector for synthetic hPDI1 and synthetic hGRP78 was designated as onaP18108.
(4) Construction of Coexpression and Constitutive Expression Vector for Synthetic hPDI Gene, Synthetic hERO1-Lα gene, and Synthetic hGRP78 Gene
A coexpression vector for synthetic hPDI and synthetic hERO1-Lα via expression regulation by GAP promoter and for synthetic hGRP78 via expression regulation by PGK promoter was constructed in the following manner.
The PGK terminator region was recovered from the pOU1/Kar2-PptK plasmid constructed in Example 4 with the use of the EcoT22I and KpnI restriction enzymes. Also, the synthetic hGRP78 region was recovered from the onaP17408 plasmid with the use of the SalI and EcoT22I restriction enzymes. Three fragments; i.e., the pUC119 plasmid (Takara Bio, TKR-3319) prepared with the aid of the SalI and KpnI restriction enzymes, the PGK terminator region recovered via digestion with the EcoT22I and KpnI restriction enzymes, and the synthetic hGRP78 region recovered via digestion with the SalI and EcoT22I restriction enzymes, were ligated to each other to construct pUC119/synthetic hGRP78+PGKt. Subsequently, the PGK promoter region recovered from pOU1/Kar2-PptK with the aid of the KpnI and SalI restriction enzymes, the synthetic hGRP78 recovered from pUC119/synthetic hGRP78+PGKt with the aid of the SalI and KpnI restriction enzymes, and the pUC119 plasmid (Takara Bio, TKR-3319) prepared via digestion of the PGK terminator region with the KpnI restriction enzyme were subjected to 3-fragment ligation. Thus, pUC119/PGKp+synthetic hGRP78+PGKt was obtained. Subsequently, pUC119/PGKp+synthetic hGRP78+PGKt was digested with the KpnI restriction enzyme, a fragment containing the PGK promoter-synthetic hGRP78-PGK terminator (the synthetic hGRP78 expression cassette) was recovered, and the resultant was introduced into the KpnI restriction enzyme site of the coexpression vector (onaP18208) for synthetic hPDI and synthetic hERO1-Lα to construct a coexpression vector (onaP18508) for synthetic hPDI, synthetic hERO1-Lα, and synthetic hGRP78.
(5) Construction of Coexpression and Constitutive Expression Vector For Synthetic hPDI Gene, Synthetic hERO1-Lβ Gene, and Synthetic hGRP78 gene
A coexpression vector for synthetic hPDI and synthetic hERO1-Lβ via expression regulation by GAP promoter and for synthetic hGRP78 via expression regulation by PGK promoter was constructed in the following manner.
The pUC119/PGKp+synthetic hGRP78+PGKt constructed in (4) above was digested with the KpnI restriction enzyme, a fragment containing the PGK promoter-synthetic hGRP78-PGK terminator (the synthetic hGRP78 expression cassette) was recovered, and the fragment was introduced into the KpnI restriction enzyme site of the coexpression vector (onaP18308) for synthetic hPDI and synthetic hERO1-Lβ to construct the coexpression vector (onaP18608) for synthetic hPDI, synthetic hERO1-Lβ, and synthetic hGRP78.
(6) Construction of Coexpression and Constitutive Expression Vector for Synthetic hPDI Gene, OmERO1 Gene, and Synthetic hGRP78 Gene
A coexpression vector for synthetic hPDI and OmERO1 via expression regulation by GAP promoter and for synthetic hGRP78 via expression regulation by PGK promoter was constructed in the following manner.
The pUC119/PGKp+synthetic hGRP78+PGKt constructed in (4) above was digested with the KpnI restriction enzyme, a fragment containing the PGK promoter-synthetic hGRP78-PGK terminator (the synthetic hGRP78 expression cassette) was recovered, and the fragment was introduced into the KpnI restriction enzyme site of the coexpression vector (onaP11107) for synthetic hPDI and OmERO1 to construct the coexpression vector (onaP18708) for synthetic hPDI, OmERO1, and synthetic hGRP78.
Example 23
Preparation of Chaperone-Introduced Yeast Strain (O. minuta)
All the constitutive expression vectors for chaperone genes constructed in Example 21 and in Example 22 were digested with the NotI restriction enzyme and then introduced into the O. minuta YK5 strain (Δoch1Δyps1Δura3Δade1: Ogataea minuta protease YPS1 gene-deficient strain) via electroporation. Electroporation was carried out under the conditions described in WO 2003/091431. After electroporation, the transformant was applied on Casamino-U agar medium that had been sterilized via steaming under pressure (6.7 g/l of Yeast Nitrogen Base without amino acids, 0.5 g/l of casamino acid, 20 g/l of glucose, 20 mg/l of L-tryptophan, 20 mg/l of adenine, and 20 g/l of Bacto agar), and the transformant was allowed to grow at 30° C. for approximately 2 to 3 days. The grown transformant was allowed to grow again on Casamino-U agar medium and a transformant into which chaperone, with its expression being regulated by the GAP promoter, had been introduced was selected via colony PCR. Part of yeast that had grown on Casamino-U agar medium was suspended in 10 μl of 0.25% SDS solution, 90 μl of sterilized water was added, and yeast cells were then removed via centrifugation at 2,700×g and 4° C. for 5 minutes. The obtained supernatant was designated as a DNA solution. A strain in which amplification was observed with the use of the GAPpforS-F primer designed within the GAP promoter sequence (5′-GATCTCAGGCCGAGTCAAGAC-3′: SEQ ID NO: 76) and the primer shown below was designated as a strain into which a constitutive expression vector for chaperone had been introduced. Since synthetic hGRP78 was expressed with the use of the PGK promoter when constructing a coexpression vector for three chaperone genes, introduction of the PGK promoter-synthetic hGRP78-PGK terminator expression cassette was confirmed with the use of the PGKpforS-F primer that was designed within the PGK promoter sequence (5′-TAACGCCGCATAGAACTAGC-3′: SEQ ID NO: 77) and the G78-EcoT-R primer (5′-ATGCATTTACAACTCGTCC-3′: SEQ ID NO: 154).
(p10) Primer used for confirming introduction of synthetic hPDI ShPDI-ttaR: 5′-GATGCATTTACAACTCGTCCTTAAC-3′ (SEQ ID NO: 78)
(p16) Primer used for confirming introduction of synthetic hERO1-Lα E1La-EcoT-R: 5′-ATGCATTTAGTGGATGTTTTG-3′ (SEQ ID NO: 155)
(p17) Primer used for confirming introduction of synthetic hERO1-Lβ E1Lb-EcoT-R: 5′-ATGCATTTATCTGGAGTGTTG-3′ (SEQ ID NO: 156)
(p18) Primers used for confirming introduction of synthetic hPDI+ synthetic hERO1-Lα
ShPDI-ttaR: 5′-GATGCATTTACAACTCGTCCTTAAC-3′ (SEQ ID NO: 78) and E1La-EcoT-R: 5′-ATGCATTTAGTGGATGTTTTG-3′ (SEQ ID NO: 155)
(p19) Primers used for confirming introduction of synthetic hPDI and synthetic hERO1-Lβ
ShPDI-ttaR: 5′-GATGCATTTACAACTCGTCCTTAAC-3′ (SEQ ID NO: 78) and E1Lb-EcoT-R: 5′-ATGCATTTATCTGGAGTGTTG-3′ (SEQ ID NO: 156)
(p20) Primers used for confirming introduction of synthetic hPDI+ synthetic hGRP78 ShPDI-ttaR: 5′-GATGCATTTACAACTCGTCCTTAAC-3′ (SEQ ID NO: 78) and G78-EcoT-R: 5′-ATGCATTTACAACTCGTCC-3′ (SEQ ID NO: 154)
(p21) Primers used for confirming introduction of synthetic hPDI, synthetic hERO1-Lα, and synthetic hGRP78
ShPDI-ttaR: 5′-GATGCATTTACAACTCGTCCTTAAC-3′ (SEQ ID NO: 78), E1La-EcoT-R: 5′-ATGCATTTAGTGGATGTTTTG-3′ (SEQ ID NO: 155), and G78-EcoT-R: 5′-ATGCATTTACAACTCGTCC-3′ (SEQ ID NO: 154)
(p22) Primers used for confirming introduction of synthetic hPDI, synthetic hERO1-Lβ, and synthetic hGRP78
ShPDI-ttaR: 5′-GATGCATTTACAACTCGTCCTTAAC-3′ (SEQ ID NO: 78), E1Lb-EcoT-R: 5′-ATGCATTTATCTGGAGTGTTG-3′ (SEQ ID NO: 156), and G78-EcoT-R: 5′-ATGCATTTACAACTCGTCC-3′ (SEQ ID NO: 154)
(p23) Primers used for confirming introduction of synthetic hPDI, OmERO1, and synthetic hGRP78
ShPDI-ttaR: 5′-GATGCATTTACAACTCGTCCTTAAC-3′ (SEQ ID NO: 78), OMEROT22I: 5′-GATGCATTTATAGCTCCAAACGATACAG-3′ (SEQ ID NO: 45), and G78-EcoT-R: 5′-ATGCATTTACAACTCGTCC-3′ (SEQ ID NO: 154)
PCR was carried out using TaKaRa LA TaqTM with GC Buffer (Takara Bio, RR02AG) at 94° C. for 30 seconds, 55° C. for 30 seconds, and 72° C. for 60 to 180 seconds to amplify the target fragment, and this cycle was repeated 30 times. The transformants below in which amplification was observed were each designated as a constitutive expression strain for chaperone.
(E17) Constitutive expression strain for synthetic hERO1-Lα; the ona56407 strain
(E18) Constitutive expression strain for synthetic hERO1-Lβ; the ona56907 strain
(E19) Constitutive expression strain for synthetic hGRP78; the ona57007 strain
(E20) Constitutive expression strain for synthetic hPDI and synthetic hERO1-Lα; the ona65708 strain
(E21) Constitutive expression strain for synthetic hPDI and synthetic hERO1-Lβ; the ona66008 strain
(E22) Constitutive expression strain for synthetic hPDI and synthetic hGRP78; the ona66108 strain
(E23) Constitutive expression strain for synthetic hPDI, synthetic hERO1-Lα, and synthetic hGRP78; the ona68708 strain
(E24) Constitutive expression strain for synthetic hPDI, synthetic hERO1-Lβ, and synthetic hGRP78; the ona68908 strain
(E25) Constitutive expression strain for synthetic hPDI, OmERO1, and synthetic hGRP78; the ona69108 strain
Example 24
Construction of Yeast Strain (O. minuta) Producing Antibody that Constitutively Expresses Chaperone
The expression vector for the anti-TRAIL receptor antibody gene (WO 2001/083560) constructed in Example 2 was introduced into a strain that constitutively expresses a single chaperone gene, a strain that constitutively expresses two types of chaperone genes, and a strain that constitutively expresses three types of chaperone genes bred in Example 23, respectively, via electroporation. As an antibody heavy chain, 1 μg of onaP02706 digested with the Sse8387I restriction enzyme was used. As an antibody light chain, 1 μg of onaP03106 digested with the NotI restriction enzyme was used. After electroporation, the transformant was applied on Casamino-U-A agar medium to which ZeocinTM (Invitrogen, R250-01) had been added at a concentration of 100 μg/ml (6.7 g/l of Yeast Nitrogen Base without amino acids, 0.5 g/l of casamino acid, 20 g/l of glucose, 20 mg/l of L-tryptophan, and 20 g/l of Bacto agar), and the transformant was allowed to grow at 30° C. for approximately 2 to 3 days. The grown transformant was allowed to grow again on Casamino-U-A agar medium to which ZeocinTM (Invitrogen, R250-01) had been added at a concentration of 100 μg/ml of agar medium, a strain that secretes and produces an antibody was screened for. The ona77108 strain was selected as the strain constitutively expressing synthetic hERO1-Lα and having the antibody gene introduced therein, the ona62508 strain was selected as the strain as the strain constitutively expressing synthetic hERO1-Lβ and having the antibody gene introduced therein, the ona61808 strain was selected as the strain constitutively expressing synthetic hGRP78 and having the antibody gene introduced therein, the ona77408 strain was selected as the strain constitutively expressing synthetic hPDI/synthetic hERO1-Lα and having the antibody gene introduced therein, the ona77808 strain was selected as the strain constitutively expressing synthetic hPDI/synthetic hERO1-Lβ and having the antibody gene introduced therein, the ona78208 strain was selected as the strain constitutively expressing synthetic hPDI/synthetic hGRP78 and having the antibody gene introduced therein, the ona78808 strain was selected as the strain constitutively expressing synthetic hPDI/synthetic hERO1-Lα/synthetic hGRP78 and having the antibody gene introduced therein, the ona79608 strain was selected as the strain constitutively expressing synthetic hPDI/synthetic hERO1-Lβ/synthetic hGRP78 and having the antibody gene introduced therein, and the ona80308 strain was selected as the strain constitutively expressing synthetic hPDI/OmERO1/synthetic hGRP78 and having the antibody gene introduced therein.
Example 25
Effects of Chaperone on Antibody Secretory Production
With the use of the strain constitutively expressing human-derived chaperone and having the antibody gene introduced therein obtained in Example 24, effects of introduction of human-derived chaperone on antibody secretory production were examined. Also, effects of antibody secretory production enhancement with the addition of PMT inhibitors that were found in Example 7 were examined. As shown in FIG. 20, a control strain (i.e., the ona02306 strain) exhibited the amount of antibody secretory production of approximately 0.4 mg/l, the strain constitutively expressing synthetic hPDI and having the antibody gene introduced therein (i.e., the ona39307 strain) exhibited that of approximately 3.1 mg/l, the strain constitutively expressing hERO1-L and having the antibody gene introduced therein (i.e., the ona77108 strain) exhibited that of approximately 0.7 mg/l, the strain constitutively expressing hERO1-Lβ and having the antibody gene introduced therein (i.e., the ona62508 strain) exhibited that of approximately 1.4 mg/l, the strain constitutively expressing hGRP78 and having the antibody gene introduced therein (i.e., the ona61808 strain) exhibited that of approximately 1.0 mg/l, the strain constitutively expressing synthetic hPDI/synthetic hERO1-Lα and having the antibody gene introduced therein (i.e., the ona77408 strain) exhibited that of approximately 1.4 mg/l, the strain constitutively expressing synthetic hPDI/synthetic hERO1-Lβ and having the antibody gene introduced therein (i.e., the ona77808 strain) exhibited that of approximately 1.0 mg/l, the strain constitutively expressing synthetic hPDI/synthetic hGRP78 and having the antibody gene introduced therein (i.e., the ona78208 strain) exhibited that of approximately 2.3 mg/l, the strain constitutively expressing synthetic hPDI/synthetic hERO1-Lα/synthetic hGRP78 and having the antibody gene introduced therein (i.e., the ona78808 strain) exhibited that of approximately 0.8 mg/l, the strain constitutively expressing synthetic hPDI/synthetic hERO1-Lβ/synthetic hGRP78 and having the antibody gene introduced therein (i.e., the ona79608 strain) exhibited that of 4.1 mg/l, and the strain constitutively expressing synthetic hPDI/OmERO1/synthetic hGRP78 and having the antibody gene introduced therein (i.e., the ona80308 strain) exhibited that of approximately 5.6 mg/l. By enhancing expression of human-derived chaperone, the capacity of antibody secretory production was improved by approximately 2 to 13 times. By adding PMT inhibitors to the culture, further, all the antibody-producing strains exhibited the increased amounts of antibody secretory production, as shown in FIG. 20. In particular, the strain constitutively expressing synthetic hPDI/synthetic hERO1-Lβ/synthetic hGRP78 and having the antibody gene introduced therein (i.e., the ona79608 strain) exhibited the amount of antibody secretory production of 9.7 mg/l and the strain constitutively expressing synthetic hPDI/OmERO1/synthetic hGRP78 and having the antibody gene introduced therein (i.e., the ona80308 strain) exhibited that of approximately 12.5 mg/l. It was demonstrated that such strains exhibited the amount of antibody secretory production improved by approximately 23 times and approximately 30 times higher than that of a control culture. As shown in FIG. 21, it was confirmed via Western blot analysis following non-reducing electrophoresis that a full-length antibody (H2L2) was apparently secreted and produced. As in the case where expression of the O. minuta-derived chaperone gene(s) alone or in combination, the capacity for antibody secretory production was also improved when expression of the human-derived chaperone gene(s) was enhanced alone or in combination. By adding PMT inhibitors to the culture, further, the capacity for antibody secretory production was improved by approximately 23 to 30 times.
Example 26
Effects of Chaperone on Human Lysozyme Expression
(1) Construction of Expression Vector for Human Lysozyme
Human lysozyme (hLyz, Accession No. NM—000239) is encoded by the gene comprising 447 base pairs and comprises 148 amino acid residues. In order to efficiently express in O. minuta and in order to express in the form of a fusion protein with the secretion signal (hereafter referred to as “aMF-secreting signal”) of S. cerevisiae-derived MF alpha1 (GENBANK Accession Number; P01149) so as to efficiently secrete in O. minuta, the aMF-secretion signal-fused hLyz gene was synthesized by taking codon preference of O. minuta into consideration. An expression vector was constructed by ligating a fragment containing the ADE1 marker that is obtained by digesting pOMex4A disclosed in WO 2003/091431 with the KpnI and ApaI restriction enzymes to a fragment containing the GAP promoter and the GAP terminator obtained by digesting pOMexGP1U disclosed in WO 2003/091431 with the KpnI and ApaI restriction enzymes, and the resultant was designated as the pOMEGPA-1 vector (SalI-EcoT22I). The aMF secretory signal-fused hLyz gene recovered via digestion with the SalI and EcoT22I restriction enzymes was introduced into pOMEGPA-1 (SalI-EcoT22I) prepared with the aid of the SalI and EcoT22I restriction enzymes to construct an expression vector for the aMF secretory signal-fused hLyz gene (onaP21808).
(2) Construction of Yeast Strain (O. minuta) Producing aMF Secretory Signal-Fused hLyz
The expression vector for the aMF secretory signal-fused hLyz gene, onaP21808, constructed in (1) was introduced into the ona01206 strain complementing a ura3 deletion mutation of the O. minuta YK5 strain constructed in Example 6, a strain constitutively expressing chaperone combination; i.e., OmPDI1, OmERO1, and OmKar2, of the O. minuta YK5 strain bred in Example 5 (i.e., the ona44607 strain), and a strain constitutively expressing human-derived chaperone combination; i.e., synthetic hPDI, synthetic hERO1-Lβ, and synthetic hGRP78, bred in Example 23 (i.e., the ona68908 strain), respectively, via electroporation. With the use of 1 μg of onaP21808 digested with the NotI restriction enzyme, the transformants were applied on Casamino-U-A agar medium (6.7 g/l of Yeast Nitrogen Base without amino acids, 0.5 g/l of casamino acid, 20 g/l of glucose, 20 mg/l of L-tryptophan, and 20 g/l of Bacto agar) after electroporation, and the transformants were allowed to grow at 30° C. for approximately 2 to 3 days. The grown transformants were allowed to grow again on Casamino-U-A agar medium and then cultured.
(3) Effects of Chaperone on Secretory Production of Human Lysozyme
The expression vector for the aMF secretory signal-fused hLyz gene, onaP21808, was introduced into the ona01206 strain into which no chaperone combination was introduced, a strain that constitutively expresses a combination of O. minuta-derived chaperone genes; i.e., OmPDI1, OmERO1, and OmKar2, (i.e., the ona44607 strain), and a strain that constitutively expresses a combination of human-derived chaperone genes; i.e., synthetic hPDI, synthetic hERO1-Lβ, and synthetic hGRP78, (i.e., the ona68908 strain). Three clones were arbitrarily selected from the resulting colonies and cultured. With the use of 2× YP-P6-GG medium (the medium was prepared by dissolving 20 g of Difco yeast extract and 40 g of Bacto peptone in 900 ml of pure water, sterilizing the solution via steaming under pressure, and adding 100 ml of separately sterilized 10× phosphate buffer (pH 6.0) (1M KH2PO4, 0.15M (NH4)2SO4, 0.375N KOH), 10 ml of a separately sterilized 50% glucose solution, and 25 ml of separately sterilized 80% glycerine), 800 μl of 2× YP-P6-GG medium was introduced into a 96-deep well plate (Greiner, 780271), the strains were sowed with the use of a toothpick, and the top of the plate was sealed with CO2-permeable plate sealers (Greiner, 676051). After culture was carried out at an agitation speed of 310 rpm, an amplitude of 50 mm, at 30° C. for 2 days, 100 μl of 2× YP-P6-GG medium containing 20 μM rhodanine-3-acetic acid derivative 1c was added, and 100 μl of 2× YP-P6-GG medium containing 20 μM of 1c was added three days later. Yeast cells were removed from the cultured solution via centrifugation at 2,700×g and 4° C. for 5 minutes, a culture supernatant was prepared, and the resultant was designated as a sample of secreted human lysozyme.
The secreted and produced human lysozyme was evaluated via Western blot analysis and in terms of bacteriolytic activity. Western blot analysis was carried out by subjecting the secreted and produced human lysozyme to SDS-PAGE under reducing conditions, blotting the separated protein on the PVDF membrane, and performing detection using the ECL Advance Western blot detection kit (GE, RPN2135) with the use of the horseradish peroxidase-conjugated IgG fraction of polyclonal rabbit antiserum to human lysozyme (Nordic Immunological Laboratories, RAHu/Lys/PO).
Also, bacteriolytic activity was assayed in the following manner. Bacteria of M. lysodeikticus was used as a substrate, and it was suspended in 50 mM phosphate buffer to prepare a substrate solution with a concentration of 0.16 mg/ml. To 240 μl of this substrate solution, 10 μl of a culture supernatant (the secreted human lysozyme) was added, and the mixture was incubated at room temperature for 10 minutes. Thereafter, the absorbance at 450 nm was assayed. Since human lysozyme degrades a bacterial cell wall, human lysozyme exhibiting higher activity exhibits a lower absorbance. Thus, a unit of lysozyme activity was defined as the amount of an enzyme that is necessary for the absorbance at 450 nm to be lowered by 0.001 per minute. Also, the total amount of proteins in the culture supernatant was assayed using the DC protein assay kit II (Bio-Rad, 500-0112JA) using bovine serum albumin as the standard.
The results of Western blot analysis demonstrate that human lysozyme is expressed in all the tested strains (FIG. 22). Also, the strain into which O. minuta-derived chaperone genes; OmPDI1, OmERO1, and OmKar2, had been introduced or the strain into which human-derived chaperone genes; synthetic hPDI, synthetic hERO1-Lβ, and synthetic hGRP78 had been introduced were found to exhibit the capacity for secretion and production of human lysozyme improved by approximately 1.5 times or higher than that in the strain into which no chaperone had been introduced, based on the assayed bacteriolytic activity (Table 3).












TABLE 3









Total






Bacteriolytic
amount of
Specific



Chaperone

activity
protein
activity


Host
introduced
Clone
(unit/ml)
(mg/ml)
(unit/mg)







Ona01206
None
1
23.0
51
0.45


Ona01206
None
2
22.3
53
0.42


Ona01206
None
3
22.0
52
0.43


Ona44607
PEK
1
33.3
50
0.67


Ona44607
PEK
2
34.0
51
0.67


Ona44607
PEK
3
34.0
53
0.65


Ona68908
hPEβG
1
36.0
53
0.68


Ona68908
hPEβG
2
29.3
48
0.61


Ona68908
hPEβG
3
33.3
51
0.65





PEK = OmPDl1 + OmERO1 + OmKar2


hPEβG = synthetic hPDl + synthetic hERO1-Lβ + synthetic hGRP78






Example 27
Construction of Constitutive Expression Strain for Chaperone Combination in Protein Mannosyl Transferase (PMT)-Deficient Strain of O. minuta 
(1) Preparation of PMT4 Gene-Deficient Strain into which Genes of the O. minuta-Derived Chaperone Proteins (PDI1/EPO1/Kar2) Had been Introduced
(1-1) Preparation of PMT4 Gene-Deficient Vector
PCR was carried out using chromosome DNA of the O. minuta IFO10746 strain as a template, the PMT4inf5′armF primer (5′-CGGGCCCCCCCTCGAGTCTATGCTCCAAGACCT-3′: SEQ ID NO: 157), and the PMT4 inf5′armR primer (5′-TACCGTCGACCTCGATCAACAACCACTGATTCC-3′: SEQ ID NO: 158) 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 2.2 kb was recovered, the recovered fragment was introduced into rURApBKS prepared with the aid of the XhoI restriction enzyme using the In-fusion kit (Clontech, 631774), and the nucleotide sequence of the inserted DNA fragment was confirmed. The resulting plasmid was designated as PMT4K/O5armrURA3. Further, PCR was carried out using chromosome DNA of the O. minuta IFO10746 strain as a template, the PMT4 inf3′armF primer (5′-AGTTCTAGAGCGGCCATCCTATACCTGTCGTGCCT-3′: SEQ ID NO: 159), and the PMT4 inf3′armR primer (5′-ACCGCGGTGGCGGCCGCTCGTGTTGTTCCAGGTAATCC-3′: SEQ ID NO: 160) 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 2.5 kb was recovered, the recovered fragment was introduced into PMT4K/O5armrURA3 prepared with the aid of the NotI restriction enzyme using the In-Fusion PCR Cloning kit (Clontech, 631774), and the nucleotide sequence of the inserted DNA fragment was confirmed. The resulting vector was designated as PMT4K/O/rURA3.
(1-2) Preparation of O. minuta PMT4-Deficient Strain (Δoch1Δyps1Δura3Δade1Δpmt4)
The PMT4 gene-deficient vector, PMT4K/O/rURA3, constructed in (1-1) above was digested with the XhoI and NotI restriction enzymes, and the resultant was introduced into the O. minuta YK5 (Δoch1Δyps1Δura3Δade1) strain via electroporation. In order to confirm that the PMT4 gene in this strain was destroyed, the primers shown below were synthesized.









(SEQ ID NO: 161)










PMT4PCR5′armF:
5′-GGTAGAGGACCGTATGTAGC-3′



 









(SEQ ID NO: 162)










PMT4PCR5′armR:
5′-CAATGAAACGTTTCCGTAGGT-3′



 









(SEQ ID NO: 163)










PMT4PCR3′armF3:
5′-TGCGAAATCGGGCCCTCT-3′



 









(SEQ ID NO: 164)










PMT4PCR3′armR3:
5′-CCGGAGTTTGCACGGCTAC-3′






Part of the transformed yeast that had been multiplied on Casamino-U agar medium was suspended in 10 μl of a 0.25% SDS solution, 90 μl of sterilized water was added, and yeast cells were then removed via centrifugation at 2,700×g and 4° C. for 5 minutes. The obtained supernatant was designated as a DNA solution. PCR was carried out using the PMT4PCR5′armF primer (SEQ ID NO: 161) and the PMT4PCR5′armR primer (SEQ ID NO: 162) at 94° C. for 30 seconds, 55° C. for 1 minute, and 72° C. for 2 minutes, and this cycle was repeated 25 times. In the strain into which the introduced fragment was incorporated into the PMT4 locus, an amplified fragment as deduced of 5.8 kb was detected. Similarly, PCR was carried out using the PMT4PCR3′armF primer (SEQ ID NO: 163) and the PMT4PCR3′armR primer (SEQ ID NO: 164) at 94° C. for 30 seconds, 55° C. for 1 minute, and 72° C. for 3 minutes, and this cycle was repeated 25 times. In the strain into which the introduced fragment was incorporated into the PMT4 locus, an amplified fragment as deduced of 7.4 kb was detected. The selected strain was designated as the O. minuta YK41 strain (Δoch1Δyps1Δura3Δade1Δpmt4::rURA3).
(1-3) Preparation of O. minuta PMT4-Deficient Strain (Δoch1Δyps1Δura3Δade1Δpmt4) Strain into which the Genes of Chaperones Had been Introduced
The O. minuta YK41 strain (Δoch1Δyps1Δura3Δade1Δpmt4::rURA3) prepared in (1-2) above was cultured in YPDA medium and applied on 5-fluoroorotic acid (5-FOA) medium (Zymo Research, F9002) to obtain the grown uracil-requiring strain. The obtained strain was designated as the ona93608 strain (Δoch1Δyps1Δura3Δade1Δpmt4). Subsequently, the constitutive expression vector, onaP11007, for OmPDI1, OmERO1, and OmKar2 constructed in Example 4 was digested with the NotI restriction enzyme and introduced into the ona93608 strain via electroporation. The transformant was applied on Casamino-U agar medium, and the transformant was allowed to grow at 30° C. for approximately 2 to 3 days. The grown transformant was allowed to grow on Casamino-U agar medium again, and a transformant into which chaperone had been introduced was selected via colony PCR. Introduction of OmPDI1 was confirmed with the use of the GAPpforS-F primer (5′-GATCTCAGGCCGAGTCAAGAC-3′: SEQ ID NO: 76) and the OMPDI1T22I primer (5′-GATGCATTTACAACTCGTCGTGAGCCAC-3′: SEQ ID NO: 29) designed within the GAP promoter sequence, that of OmERO1 was confirmed with the use of GAPpforS-F (5′-GATCTCAGGCCGAGTCAAGAC-3′: SEQ ID NO: 76) and OMEROT22I (5′-GATGCATTTATAGCTCCAAACGATACAG-3′: SEQ ID NO: 45), and that of OmKar2 was confirmed with the use of the PGKpforS-F primer (5′-TAACGCCGCATAGAACTAGC-3′: SEQ ID NO: 77) and the OMKAR-R primer (5′-GATGCATTCACAGCTCATCATGATCCCAG-3′: SEQ ID NO: 51) designed within the PGK promoter sequence. The obtained O. minuta PMT4-deficient strain (Δoch1Δyps1Δura3Δade1Δpmt4) into which the genes of chaperones (OmPDI1/OmERO1/OmKar2) had been introduced was designated as the ona96708 strain.
(2) Preparation of the PMT5 Gene-Deficient Strain into which the Genes of O. minuta-Derived Chaperone Proteins (PDI1/EPO1/Kar2) had been Introduced
The O. minuta YK6 strain (Δoch1Δyps1Δura3Δade1Δpmt5::rURA3) disclosed in WO 2007/132949 was cultured in YPDA medium and applied on 5-fluoroorotic acid (5-FOA) medium (Zymo Research, F9002) to obtain a grown uracil-requiring strain. The obtained strain was designated as the ona64908 strain (Δoch1Δyps1Δura3Δade1Δpmt5). Subsequently, the constitutive expression vector, onaP11007, for OmPDI1, OmERO1, and OmKar2 constructed in Example 4 was digested with the NotI restriction enzyme and introduced into the ona64908 strain via electroporation. The transformant was applied on Casamino-U agar medium, and the transformant was allowed to grow at 30° C. for approximately 2 to 3 days. The grown transformant was allowed to grow on Casamino-U agar medium again, and a transformant into which chaperone had been introduced was selected via colony PCR. Introduction of OmPDI1 was confirmed with the use of the GAPpforS-F primer (5′-GATCTCAGGCCGAGTCAAGAC-3′: SEQ ID NO: 76) and the OMPDI1T22I primer (5′-GATGCATTTACAACTCGTCGTGAGCCAC-3′: SEQ ID NO: 29) designed within the GAP promoter sequence, that of OmERO1 was confirmed with the use of GAPpforS-F (5′-GATCTCAGGCCGAGTCAAGAC-3′: SEQ ID NO: 76) and OMEROT22I (5′-GATGCATTTATAGCTCCAAACGATACAG-3′: SEQ ID NO: 45), and that of OmKar2 was confirmed with the use of the PGKpforS-F primer (5′-TAACGCCGCATAGAACTAGC-3′: SEQ ID NO: 77) and the OMKAR-R primer (5′-GATGCATTCACAGCTCATCATGATCCCAG-3′: SEQ ID NO: 51) designed within the PGK promoter sequence. The obtained PMT5-deficient strain of O. minuta (Δoch1Δyps1Δura3Δade1Δpmt5) into which the genes of chaperone (OmPDI1/OmERO1/OmKar2) had been introduced was designated as the ona69308 strain.
(3) Preparation of the PMT6 Gene-Deficient Strain into which the Genes of O. minuta-Derived Chaperone Proteins (PDI1/EPO1/Kar2) had been Introduced
The O. minuta YK7 strain (Δoch1Δyps1Δura3Δade1Δpmt6::rURA3) disclosed in WO 2007/132949 was cultured in YPDA medium and applied on 5-fluoroorotic acid (5-FOA) medium (Zymo Research, F9002) to obtain a grown uracil-requiring strain. The obtained strain was designated as the ona65008 strain (Δoch1Δyps1Δura3Δade1Δpmt6). Subsequently, the constitutive expression vector, onaP11007, for OmPDI1, OmERO1, and OmKar2 constructed in Example 4 was digested with the NotI restriction enzyme and introduced into the ona65008 strain via electroporation. The transformant was applied on Casamino-U agar medium, and the transformant was allowed to grow at 30° C. for approximately 2 to 3 days. The grown transformant was allowed to grow on Casamino-U agar medium again, and a transformant into which chaperone had been introduced was selected via colony PCR. Introduction of OmPDI1 was confirmed with the use of the GAPpforS-F primer (5′-GATCTCAGGCCGAGTCAAGAC-3′: SEQ ID NO: 76) and the OMPDI1T22I primer (5′-GATGCATTTACAACTCGTCGTGAGCCAC-3′: SEQ ID NO: 29) designed within the GAP promoter sequence, that of OmERO1 was confirmed with the use of GAPpforS-F (5′-GATCTCAGGCCGAGTCAAGAC-3′: SEQ ID NO: 76) and OMEROT22I (5′-GATGCATTTATAGCTCCAAACGATACAG-3′: SEQ ID NO: 45), and that of OmKar2 was confirmed with the use of the PGKpforS-F primer (5′-TAACGCCGCATAGAACTAGC-3′: SEQ ID NO: 77) and the OMKAR-R primer (5′-GATGCATTCACAGCTCATCATGATCCCAG-3′: SEQ ID NO: 51) designed within the PGK promoter sequence. The obtained O. minuta PMT6-deficient strain (Δoch1Δyps1Δura3Δade1Δpmt6) into which the genes of chaperones (OmPDI1/OmERO1/OmKar2) had been introduced was designated as the ona69508 strain.
(4) Preparation of the PMT5/PMT6 Double-Deficient Strain into which the Genes of O. minuta-Derived Chaperone Proteins (PDI1/EPO1/Kar2) Had been Introduced
(4-1) Destruction of PMT5 Gene in PMT6 Gene-Deficient Strain
The PMT5 gene-deficient vector, PMT5K/O/rURA3, disclosed in WO 2007/132949 was digested with the HindIII restriction enzyme and it was then introduced into the ona65008 strain (Δoch1Δyps1Δura3Δade1Δpmt6) prepared in (3) above via electroporation. Whether or not the PMT5 gene was destroyed in the strain was confirmed with the use of the primer disclosed in WO 2007/132949. Part of the transformant yeast that had been multiplied on Casamino-U agar medium was suspended in 10 μl of a 0.25% SDS solution, 90 μl of sterilized water was added, and yeast cells were then removed via centrifugation at 2,700×g and 4° C. for 5 minutes. The obtained supernatant was designated as a DNA solution. PCR was carried out using the gPMT5-5 primer (5′-CGGTGACGACTTCGACTAGTCGAG-3′: SEQ ID NO: 165) and the gPMT5-2 primer (5′-CGGTGCTGTTGGCGTCGTCATGGGTG-3′: SEQ ID NO: 166) at 94° C. for 30 seconds, 55° C. for 1 minute, and 72° C. for 2 minutes, and this cycle was repeated 25 times. In the strain into which the introduced fragment was incorporated into the PMT5 locus, an amplified fragment as deduced of 4.9 kb was detected. Similarly, PCR was carried out using the gPMT5-3 primer (5′-GGCGCGTTCCAATTCCACTCTGCTG-3′: SEQ ID NO: 167) and the gPMT5-4 primer (5′-CGACGAGTCCTCTCACCAGGAGGTTG-3′: SEQ ID NO: 168) at 94° C. for 30 seconds, 55° C. for 1 minute, and 72° C. for 2 minutes, and this cycle was repeated 25 times. In the strain into which the introduced fragment was incorporated into the PMT5 locus, an amplified fragment as deduced of 4.9 kb was detected. The selected strain was designated as the O. minuta YK51 strain (Δoch1Δyps1Δura3Δade1Δpmt6Δpmt5::rURA3).
(4-2) Preparation of O. minuta PMT5/PMT6 Double-Deficient Strain (Δoch1Δyps1Δura3Δade1Δpmt5Δpmt6) into which Chaperone Had been Introduced
The O. minuta YK51 strain (Δoch1Δyps1Δura3Δade1Δpmt6Δpmt5::rURA3) prepared in (4-1) above was cultured in YPDA medium and applied on 5-fluoroorotic acid (5-FOA) medium (Zymo Research, F9002) to obtain a grown uracil-requiring strain. The obtained strain was designated as the ona15707 strain (Δoch1Δyps1Δura3Δade1Δpmt5Δpmt6). Subsequently, the constitutive expression vector, onaP11007, for OmPDI1, OmERO1, and OmKar2 constructed in Example 4 was digested with the NotI restriction enzyme and introduced into the ona15707 strain via electroporation. The transformant was applied on Casamino-U agar medium, and the transformant was allowed to grow at 30° C. for approximately 2 to 3 days. The grown transformant was allowed to grow on Casamino-U agar medium again, and a transformant into which chaperone had been introduced was selected via colony PCR. Introduction of OmPDI1 was confirmed with the use of the GAPpforS-F primer (5′-GATCTCAGGCCGAGTCAAGAC-3′: SEQ ID NO: 76) and the OMPDI1T22I primer (5′-GATGCATTTACAACTCGTCGTGAGCCAC-3′: SEQ ID NO: 29) designed within the GAP promoter sequence, that of OmERO1 was confirmed with the use of GAPpforS-F (5′-GATCTCAGGCCGAGTCAAGAC-3′: SEQ ID NO: 76) and OMEROT22I (5′-GATGCATTTATAGCTCCAAACGATACAG-3′: SEQ ID NO: 45), and that of OmKar2 was confirmed with the use of the PGKpforS-F primer (5′-TAACGCCGCATAGAACTAGC-3′: SEQ ID NO: 77) and the OMKAR-R primer (5′-GATGCATTCACAGCTCATCATGATCCCAG-3′: SEQ ID NO: 51) designed within the PGK promoter sequence. The obtained O. minuta PMT5/PMT6 double-deficient strain (Δoch1Δyps1Δura3Δade1Δpmt5Δpmt6) into which the genes of chaperones (OmPDI1/OmERO1/OmKar2) had been introduced was designated as the ona56207 strain.
(5) Construction of Antibody-Producing Yeast Strain (O. minuta) Utilizing the PMT Gene-Deficient Strain into which Genes of the O. minuta-Derived Chaperone Proteins (PDI1/EPO1/Kar2) were Introduced
The expression vectors for the anti-TRAIL receptor antibody gene (WO 2001/083560) constructed in Example 2 were introduced into the strains shown below prepared by introducing the chaperone genes (OmPDI1/OmERO1/OmKar2) into the PMT-deficient strains constructed in (1) to (4) above via electroporation.
O. minuta PMT4-deficient strain (Δoch1Δyps1Δura3Δade1Δpmt4) into which the genes of chaperones (OmPDI1/OmERO1/OmKar2) had been introduced (i.e., the ona96708 strain)
O. minuta PMT5-deficient strain (Δoch1Δyps1Δura3Δade1Δpmt5) into which the genes of chaperones (OmPDI1/OmERO1/OmKar2) had been introduced (i.e., the ona69308 strain)
O. minuta PMT6-deficient strain (Δoch1Δyps1Δura3Δade1Δpmt6) into which the genes of chaperones (OmPDI1/OmERO1/OmKar2) had been introduced (i.e., the ona69508 strain)
O. minuta PMT5/PMT6 double-deficient strain (Δoch1Δyps1Δura3Δade1Δpmt5Δpmt6) into which the genes of chaperones (OmPDI1/OmERO1/OmKar2) had been introduced (i.e., the ona56207 strain)
As an antibody heavy chain, 1 μg of onaP02706 digested with the Sse8387I restriction enzyme was used. As an antibody light chain, 1 μg of onaP03106 digested with the NotI restriction enzyme was used. After electroporation, the transformant was applied on Casamino-U-A agar medium to which ZeocinTM (Invitrogen, R250-01) had been added at a concentration of 100 μg/ml (6.7 g/l of Yeast Nitrogen Base without amino acids, 0.5 g/l of casamino acid, 20 g/l of glucose, 20 mg/l of L-tryptophan, and 20 g/l of Bacto agar), and the transformant was allowed to grow at 30° C. for approximately 2 to 3 days. The grown transformant was allowed to grow again on Casamino-U-A agar medium to which ZeocinTM (Invitrogen, R250-01) had been added at a concentration of 100 μg/ml, and a strain that secretes and produces an antibody was screened for. The ona98808 strain was selected as the O. minuta PMT4-deficient strain constitutively expressing the chaperones (OmPDI1/OmERO1/OmKar2) and having the antibody gene introduced therein; the ona74108 strain was selected as the O. minuta PMT5-deficient strain constitutively expressing the chaperones (OmPDI1/OmERO1/OmKar2) and having the antibody gene introduced therein; the ona74608 strain was selected as the O. minuta PMT6-deficient strain constitutively expressing the chaperones (OmPDI1/OmERO1/OmKar2) and having the antibody gene introduced therein; and the ona67408 strain was selected as the O. minuta PMT5/PMT6 double-deficient strain constitutively expressing the chaperones (OmPDI1/OmERO1/OmKar2) and having the antibody gene introduced therein.
Example 28
Confirmation of capacity for antibody production of antibody-producing yeast strain (O. minuta) prepared by introducing genes of O. minuta-derived chaperone proteins (PDI1/EPO1/Kar2) into the Protein Mannosyl Transferase (PMT) gene-deficient strain of O. minuta 
The capacity for antibody secretory production of the PMT-deficient strain constitutively expressing the chaperones (OmPDI1/OmERO1/OmKar2) and having the antibody gene introduced therein constructed in Example 27 (5), was confirmed. Also, effects of antibody secretory production enhancement with the addition of PMT inhibitors that were found in Example 7 were examined.
The antibody-producing strain was cultured in the following manner. With the use of 2× YP-P6-GG medium (the medium was prepared by dissolving 20 g of Difco yeast extract and 40 g of Bacto peptone in 900 ml of pure water, sterilizing the solution via steaming under pressure, and adding 100 ml of separately sterilized 10× phosphate buffer (pH 6.0) (1M KH2PO4, 0.15M (NH4)2SO4, 0.375N KOH), 10 ml of a separately sterilized 50% glucose solution, and 25 ml of separately sterilized 80% glycerine), 800 μl of 2× YP-P6-GG medium was introduced into a 96-deep well plate (Greiner, 780271), the strains were sowed with the use of a toothpick, and the top of the plate was sealed with CO2-permeable plate sealers (Greiner, 676051). Culture was carried out at an agitation speed of 310 rpm, an amplitude of 50 mm, at 30° C., and 100 μl each of 2× YP-P6-GG medium was added 2 and 3 days after the initiation of culture. Culture involving the addition of PMT inhibitors was carried out with the use of 2× YP-P6-GG medium (the medium was prepared by dissolving 20 g of Difco yeast extract and 40 g of Bacto peptone in 900 ml of pure water, sterilizing the solution via steaming under pressure, and adding 100 ml of separately sterilized 10× phosphate buffer (pH 6.0) (1M KH2PO4, 0.15M (NH4)2SO4, 0.375N KOH), 10 ml of a separately sterilized 50% glucose solution, and 25 ml of separately sterilized 80% glycerine), 800 μl of 2× YP-P6-GG medium was introduced into a 96-deep well plate (Greiner, 780271), the strains were sowed with the use of a toothpick, and the top of the plate was sealed with CO2-permeable plate sealers (Greiner, 676051). Culture involving the addition of the rhodanine-3-acetic acid derivative 1c was first carried out at an agitation speed of 310 rpm, an amplitude of 50 mm, at 30° C. for 2 days, 100 μl of 2× YP-P6-GG medium containing 20 μM 1c was added, and 100 μl of 2× YP-P6-GG medium containing 20 μM 1c was further added on day 3.
Secretion and production of an antibody were confirmed via Sandwich ELISA or Western blotting. Yeast cells were removed from the culture product via centrifugation at 2,700×g and 4° C. for 5 minutes, and the resulting culture supernatant was designated as a sample of secreted antibody. 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, the sample of secreted antibody 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, 04-10-20) and the ABTS peroxidase substrate (KPL, 50-66-01). Western blotting was carried out as follows. After the protein was subjected to SDS-PAGE under reducing and non-reducing conditions, the separated protein was blotted to a PVDF membrane, and the antibody heavy chain and light chain were detected using Anti-human IgG (γ-chain specific) (Sigma, I-3382) and Goat anti human kappa b&f affinity purified (Bethyl, A-80-115A) as the primary antibodies. Peroxidase conjugated affinity purified anti-goat IgG (H&L), rabbit, Rockland, #605-4313) was used as the secondary antibody. Detection was carried out using the ECL Advance Western blotting detection kit (GE, RPN2135).
As shown in FIG. 23, a control strain (i.e., the ona02306 strain) exhibited the amount of antibody secretory production of approximately 0.6 mg/l; the strain constitutively expressing chaperones (OmPDI1/OmERO1/OmKar2) and having the antibody gene introduced therein (i.e., the ona48707 strain) exhibited that of approximately 8.2 mg/l; the O. minuta PMT4-deficient strain constitutively expressing chaperones (OmPDI1/OmERO1/OmKar2) and having the antibody gene introduced therein (i.e., the ona98808 strain) exhibited that of approximately 0.4 mg/l; the O. minuta PMT5-deficient strain constitutively expressing chaperones (OmPDI1/OmERO1/OmKar2) and having the antibody gene introduced therein (i.e., the ona74108 strain) exhibited that of approximately 3.8 mg/l; the O. minuta PMT6-deficient strain constitutively expressing chaperones (OmPDI1/OmERO1/OmKar2) and having the antibody gene introduced therein (i.e., the ona74608 strain) exhibited that of approximately 8 mg/l; and the O. minuta PMT5/PMT6 double-deficient strain constitutively expressing chaperones (OmPDI1/OmERO1/OmKar2) and having the gene of an antibody introduced therein (i.e., the ona67408 strain) exhibited that of approximately 8.3 mg/l. Except for the PMT4 gene-deficient strain, enhanced chaperone expression was effective for a single- or double-deficient strain of the PMT gene, and the capacity for antibody secretory production was improved by approximately 5 to 14 times greater than that of a control strain.
Further, addition of PMT inhibitors, rhodanine, to culture resulted in an improvement in the amount of antibody secretory production by approximately 20 times to approximately 33 times in all the antibody-producing strains, as shown in FIG. 23. In the case of the O. minuta PMT5-deficient strain constitutively expressing chaperones (OmPDI1/OmERO1/OmKar2) and having the antibody gene introduced therein (i.e., the ona74108 strain) and the O. minuta PMT6-deficient strain constitutively expressing chaperones (OmPDI1/OmERO1/OmKar2) and having the antibody gene introduced therein (i.e., the ona74608 strain), the amount of antibody secretory production was reduced to approximately 75% that of the strain constitutively expressing chaperones (OmPDI1/OmERO1/OmKar2) and having the antibody gene introduced therein (i.e., the ona48707 strain). In the case of the O. minuta PMT5/PMT6 double-deficient strain constitutively expressing chaperones (OmPDI1/OmERO1/OmKar2) and having the antibody gene introduced therein (i.e., the ona67408 strain), the amount of antibody secretory production thereof was reduced to approximately 60% that of the strain constitutively expressing chaperones (OmPDI1/OmERO1/OmKar2) and having the antibody gene introduced therein (i.e., the ona48707 strain).
As described above, the O. minuta PMT4-deficient strain constitutively expresses chaperones (OmPDI1/OmERO1/OmKar2) and having the antibody gene introduced therein (i.e., the ona98808 strain), which exhibited the amount of production as low as approximately 0.4 mg/l, underwent abnormal growth and division resulting from PMT4 gene deficiency as shown in FIG. 24 and in FIG. 25. Regarding the O. minuta PMT4-deficient strain constitutively expressing chaperones (OmPDI1/OmERO1/OmKar2) and having the antibody gene introduced therein (i.e., the ona98808 strain), the amount of an inhibitor of PMT activity, rhodanine-3-acetic acid derivative 1c added, was examined. Culture was carried out by introducing 800 μl of 2× YP-P6-GG medium to a 96-deep well plate (Greiner, 780271), 100 μl each of medium prepared while adjusting the concentration of the rhodanine-3-acetic acid derivative 1c at 1.25, 2.5, 5, 10, 20, or 40 μM with the aid of 2× YP-P6-GG medium was added 2 and 3 days after the initiation of culture (final concentration: 0.25, 0.5, 1, 2, 4, or 8 μM). As a result, the amount of production was improved by approximately 13% when 2× YP-P6-GG medium containing 0.25 μM 1c (final concentration) was added, as shown in FIG. 26. Concentration of the inhibitor of PMT activity, rhodanine-3-acetic acid derivative 1c, to be added to the O. minuta PMT4-deficient strain constitutively expressing chaperones (OmPDI1/OmERO1/OmKar2) and having the antibody gene introduced therein (i.e., the ona98808 strain), was determined to be approximately 1/16 of other strains.
Example 29
Purification of Antibody Produced by Antibody-Producing Yeast Strain (O. minuta) Prepared by Introducing the Genes of the O. minuta-Derived Chaperone Proteins (PDI1/EPO1/Kar2) into the PMT Gene-Deficient Strain
As described in Example 28, it was found that destruction of the PMT gene of O. minuta would lower the capacity for antibody secretory production to some extent and would significantly influence the morphology of O. minuta. This strongly suggests that suppression of O sugar chain addition via suppression of PMT activity of O. minuta significantly influences physiological functions of O. minuta. Thus, evaluation of biological activity (i.e., cytotoxic activity) of antibodies produced by such antibody-producing strains is considered to lead to discovery of optimal conditions for combinations of PMT mutation, constitutive expression of chaperone proteins (PDI1/EPO1/Kar2), and addition of an inhibitor of PMT activity (rhodanine-3-acetic acid derivative 1c).
(1) Culture of Antibody-Producing Yeast Strain (O. minuta) Prepared by Introducing the Genes of the O. minuta-Derived Chaperone Proteins (PDI1/EPO1/Kar2) into the PMT Gene-Deficient Strain
The ona02306 strain was selected as a control strain (i.e., a strain into which no chaperones are introduced, but the antibody gene is introduced); the ona48707 strain was selected as a strain constitutively expressing chaperones (OmPDI1/OmERO1/OmKar2) and having the antibody gene introduced therein, the ona98808 strain was selected as the O. minuta PMT4-deficient strain constitutively expressing chaperones (OmPDI1/OmERO1/OmKar2) and having the antibody gene introduced therein, the ona74108 strain was selected as the O. minuta PMT5-deficient strain constitutively expressing chaperones (OmPDI1/OmERO1/OmKar2) and having the antibody gene introduced therein, the ona74608 strain was selected as the O. minuta PMT6-deficient strain constitutively expressing chaperones (OmPDI1/OmERO1/OmKar2) and having the antibody gene introduced therein, and the ona67408 strain was selected as the O. minuta PMT5/PMT6 double-deficient strain constitutively expressing chaperones (OmPDI1/OmERO1/OmKar2) and having the antibody gene introduced therein.
A 100-ml Erlenmeyer flask comprising 20 ml of Casamino-U-A medium prepared by adding ZeocinTM (Invitrogen, R250-01) at a concentration of 50 μg/ml to Casamino-U-A medium (6.7 g/l of Yeast Nitrogen Base without amino acids, 0.5 g/l of casamino acid, 20 g/l of glucose, and 20 mg/l of L-tryptophan) was subjected to seed culture at an agitation speed of 210 rpm, an amplitude of 75 mm, at 30° C. for 2 days. Subsequently, 20 to 30 ml of the seed culture solution was introduced into a 2-1 Erlenmeyer flask containing 400 ml of 2× YP-P6-GG medium (the medium was prepared by dissolving 20 g of Difco yeast extract and 40 g of Bacto peptone in 900 ml of pure water, sterilizing the solution via steaming under pressure, and adding 100 ml of separately sterilized 10× phosphate buffer (pH 6.0) (1M KH2PO4, 0.15M (NH4)2SO4, 0.375N KOH), 10 ml of a separately sterilized 50% glucose solution, and 25 ml of separately sterilized 80% glycerine), and culture was carried out at an agitation speed of 210 rpm, an amplitude of 75 mm, at 30° C. After the OD 600 value exceeded 10, an inhibitor of PMT activity (rhodanine-3-acetic acid derivative 1c) (stock solution concentration: 40 mM) was added in amounts of 0.2 μM each as the OD600 value increased by 1. Only in the case of the O. minuta PMT4-deficient strain constitutively expressing chaperones (OmPDI1/OmERO1/OmKar2) and having the antibody gene introduced therein (i.e., the ona98808 strain), the inhibitor of PMT activity was added in amounts of 0.0125 μM each.
(2) Purification of Antibody Produced from O. minuta 
Yeast cells were removed from the culture solution via centrifugation at 13,000×g, 4° C., for 30 minutes, and the culture supernatant was designated as a sample of secreted antibody. The sample was concentrated using Pall Filtron CentrimateTM (10K Omega Centrimate medium screen, Pall) and then purified with the use of the AKTA purifier (GE). At the outset, the concentrated antibody sample was adsorbed to the HiTrap Mabselect SuRe (GE, 11-0034-94), which had been equilibrated with sodium phosphate buffer (20 mM sodium phosphate, 300 mM sodium chloride, pH 7.2), and the sample was eluted with the use of ImmunoPure IgG Elution Buffer (Pierce, 21009). The eluted fraction was immediately neutralized with the addition of 1M Tris-HCl buffer (pH 9) in an amount of one tenth of the elution fraction, and an equivalent amount of sodium phosphate buffer (100 mM sodium phosphate, 150 mM sodium chloride, pH 7.2) was then added. Subsequently, the fraction eluted with the use of HiTrap Mabselect SuRe was adsorbed to Protein L Cartridge (Pierce, #89929), which had been equilibrated with sodium phosphate buffer (100 mM sodium phosphate, 150 mM sodium chloride, pH 7.2), and the sample was eluted with the use of ImmunoPure IgG Elution Buffer (Pierce, 21009). The eluted Protein L Cartridge-purified fraction was subjected to gel filtration with the use of Superdex200 10/30 GL (GE, 17-5175-01), which had been equilibrated with sodium phosphate buffer (10 mM sodium phosphate, 150 mM sodium chloride, pH 7.2) to purify various antibodies produced from O. minuta. The purified antibodies were analyzed via size exclusion chromatography (SEC)-HPLC using PROTEIN KW403-4F (4.6 (i.d.)×300 mm) (Showa Denko K.K., F6989202). Sodium phosphate buffer (30 mM sodium phosphate, 300 mM sodium chloride, pH 6.7) was used as a mobile phase. FIG. 27 shows a pattern of elution via SEC-HPLC of the purified antibodies. While the ona02306 strain (the control strain containing no chaperones and having the antibody gene introduced therein) and the PMT4-deficient strain constitutively expressing chaperones (OmPDI1/OmERO1/OmKar2) and having antibody gene introduced therein (i.e., the ona98808) produced broad chromatograms, the strain constitutively expressing chaperones (OmPDI1/OmERO1/OmKar2) and having the antibody gene introduced therein (i.e., the ona48707 strain), the PMT5-deficient strain constitutively expressing chaperones (OmPDI1/OmERO1/OmKar2) and having the antibody gene introduced therein (i.e., the ona74108 strain), the PMT6-deficient strain constitutively expressing chaperones (OmPDI1/OmERO1/OmKar2) and having the antibody gene introduced therein (i.e., the ona74608 strain), and the PMT5/PMT6 double-deficient strain constitutively expressing chaperones (OmPDI1/OmERO1/OmKar2) and having the antibody gene introduced therein (i.e., the ona67408 strain) produced sharp chromatograms.
The results of reducing/non-reducing SDS-PAGE followed by Western blot analysis demonstrate that the heterotetramer of the antibody heavy chain and light chain was not clearly detected in the ona02306 strain (the control strain containing no chaperones and having the antibody gene introduced therein) and the PMT4-deficient strain constitutively expressing chaperones (OmPDI1/OmERO1/OmKar2) and having antibody gene introduced therein (i.e., the ona98808) under non-reducing conditions as shown in FIG. 28. On the other hand, the heterotetramer of the antibody heavy chain and light chain was clearly detected in the strain constitutively expressing chaperones (OmPDI1/OmERO1/OmKar2) and having the antibody gene introduced therein (i.e., the ona48707 strain), the PMT5-deficient strain constitutively expressing chaperones (OmPDI1/OmERO1/OmKar2) and having the antibody gene introduced therein (i.e., the ona74108 strain), the PMT6-deficient strain constitutively expressing chaperones (OmPDI1/OmERO1/OmKar2) and having the antibody gene introduced therein (i.e., the ona74608 strain), and the PMT5/PMT6 double-deficient strain constitutively expressing chaperones (OmPDI1/OmERO1/OmKar2) and having the antibody gene introduced therein (i.e., the ona67408 strain). Enhancement of chaperone (OmPDI1/OmERO1/OmKar2) expression was found to accelerate formation of an assembly; i.e., the heterotetramer of the antibody heavy chain and light chain.
(3) Cytotoxic Activity of Antibodies Produced from O. minuta 
(3-1) Measurement of Amount of Purified Antibody Protein
The amount of the purified antibody protein was measured by employing bovine serum albumin as the standard and using the DC protein assay kit II (Bio-Rad, 500-0112JA).
(3-2) Measurement of Cytotoxic Activity of Purified Antibody
Cytotoxic activity of the anti-TRAIL receptor antibody was measured by the cross-link method using secondary antibodies as described below. An anti-TRAIL receptor antibody produced with the use of a mammalian cell was used as a control sample. The goat affinity purified antibody to human IgG Fc (MP Biomedicals, #55071) was used as a secondary antibody. The concentration of the secondary antibody was adjusted at 1 mg/ml with the use of cell culture medium (RPMI 1640 medium containing 10% serum, GIBCO, 11875), and the cell culture medium containing the secondary antibody thus prepared was used to prepare a specimen at a concentration of 2,000 ng/ml. Further, a dilution series of the prepared specimen was prepared (200 ng/ml, 20 ng/ml, 2 ng/ml, and 0.2 ng/ml; 10-fold diluted) and fractionated on the Corning 96 well plate (costar, #3598) at 50 μl/well. A Jurkat cell suspension (1×105 cells/ml) was sowed on the plate to which the specimen had been added at 50 μl/well. ATP derived from cells having metabolic activity was quantified 72 hours after the initiation of culture with the use of the ATP detection kit (CellTiter-GloTM Luminescent Cell Viability Assay, Promega, #G7571), cell survival of the untreated group was designated as 100%, and the viable cell count in each treated group was determined. As shown in FIG. 29, whereas the anti-TRAIL antibody produced from animal cells exhibited IC50 of 7 ng/ml, the antibody produced from the ona02306 strain (the control strain containing no chaperones and having the antibody gene introduced therein) exhibited IC50 of 77.3 ng/ml; the antibody produced from the ona48707 strain constitutively expressing chaperones (OmPDI1/OmERO1/OmKar2) and having the antibody gene introduced therein exhibited IC50 of 28.5 ng/ml; the antibody produced from the ona74108 strain (the PMT5-deficient strain constitutively expressing chaperones (OmPDI1/OmERO1/OmKar2) and having the antibody gene introduced therein) exhibited IC50 of 26.6 ng/ml; the antibody produced from the ona74608 (the PMT6-deficient strain constitutively expressing chaperones (OmPDI1/OmERO1/OmKar2) and having the antibody gene introduced therein) exhibited IC50 of 16.8 ng/ml; the antibody produced from the ona67408 strain (the PMT5/PMT6 double-deficient strain constitutively expressing chaperones (OmPDI1/OmERO1/OmKar2) and having the antibody gene introduced therein) exhibited IC50 of 21.8 ng/ml. As shown in FIG. 30, whereas the anti-TRAIL antibody produced from animal cells exhibited IC50 of 6.7 ng/ml, the antibody produced from the ona48707 strain constitutively expressing chaperones (OmPDI1/OmERO1/OmKar2) and having the antibody gene introduced therein exhibited IC50 of 25.3 ng/ml; the antibody produced from the ona74608 (the PMT6-deficient strain constitutively expressing chaperones (OmPDI1/OmERO1/OmKar2) and having the antibody gene introduced therein) exhibited IC50 of 21.6 ng/ml; the antibody produced from the ona98808 strain (the PMT4-deficient strain constitutively expressing chaperones (OmPDI1/OmERO1/OmKar2) and having the antibody gene introduced therein) exhibited IC50 of 28.2 ng/ml.
Cytotoxic activity of the antibody produced from the ona02306 strain (the control strain containing no chaperones and having the antibody gene introduced therein) was as small as one eleventh that of the anti-TRAIL receptor antibody produced from animal cells. The antibody produced by the ona48707 strain constitutively expressing chaperones (OmPDI1/OmERO1/OmKar2) and having the antibody gene introduced therein exhibited cytotoxic activity of 1/4.1 times that of the anti-TRAIL receptor antibody produced from animal cells and 2.7 times that of the control strain. While all of the PMT-deficient strains constitutively expressing chaperones (OmPDI1/OmERO1/OmKar2) and having the antibody gene introduced therein exhibited higher cytotoxic activity than that of the control strain, the antibody produced from the PMT6-deficient strain constitutively expressing chaperones (OmPDI1/OmERO1/OmKar2) and having the antibody gene introduced therein (i.e., the ona74608 strain) exhibited the highest cytotoxic activity, which was 1/2.4 times that of the anti-TRAIL receptor antibody produced from animal cells and 4.6 times higher than that of the control strain. The results demonstrate that an antibody produced by introducing chaperones (OmPDI1/OmERO1/OmKar2) into the PMT6 gene-deficient strain of O. minuta with the addition of the PMT inhibitors (i.e., the rhodanine-3-acetic acid derivative 1c) exhibited the highest biological activity.
All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.
INDUSTRIAL APPLICABILITY
According to the present invention, introduction of chaperone genes into host cells enables high-level secretion and production of proteins having complicated structures (e.g., antibodies) that are correctly folded in host cells, as well as normal proteins. By employing chaperone gene introduction in combination with suppression of O sugar chain addition inherent to yeast, synergetic effects can be attained regarding high-level secretion and production of proteins.

## Claims

1. A transformed host cell which is transformed with a nucleic acid comprising an isolated polynucleotide encoding a protein disulfide isomerase (PDI), wherein said isolated polynucleotide is selected from the group consisting of (a) and (b):
(a) an isolated polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1; and
(b) an isolated polynucleotide comprising a nucleotide sequence having at least 95% nucleotide sequence identity to the nucleotide sequence of SEQ ID NO: 1, wherein said nucleotide sequence having at least 95% nucleotide sequence identity to the nucleotide sequence of SEQ ID NO: 1 encodes a protein having an activity of accelerating increasing secretion of a foreign protein.

2. The transformed host cell of claim 1, wherein said transformed host cell further comprises an isolated polynucleotide selected from (i) and (ii):
(i) an isolated polynucleotide comprising the nucleotide sequence of any one of SEQ ID NOs: 3, 5, 7, 9, 11 and 13; and
(ii) an isolated polynucleotide comprising a nucleotide sequence having at least 95% nucleotide sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 3, 5, 7, 9, 11 and 13, wherein said nucleotide sequence having at least 95% nucleotide sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 3, 5, 7, 9, 11 and 13 encodes a protein having an activity of increasing secretion of a foreign protein.

3. A transformed host cell which is transformed with a nucleic acid comprising an isolated polynucleotide encoding a protein disulfide isomerase (PDI), wherein said isolated polynucleotide encodes a polypeptide selected from the group consisting of (a) and (b):
(a) a polypeptide comprising the amino acid sequence of SEQ ID NO: 2; and
(b) a polypeptide comprising an amino acid sequence having at least 95% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 2, wherein said polypeptide comprising an amino acid sequence having at least 95% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 2 has an activity of increasing secretion of a foreign protein.

4. The transformed host cell of claim 3, wherein said transformed host cell further comprises an isolated polynucleotide encoding a polypeptide selected from (i) and (ii):
(i) a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 4, 6, 8, 10, 12 and 14; and
(ii) a polypeptide comprising an amino acid sequence having at least 95% amino acid sequence identity to the amino acid sequence of any one of SEQ ID NOs: 4, 6, 8, 10, 12 and 14, wherein said polypeptide comprising an amino acid sequence having at least 95% amino acid sequence identity to the amino acid sequence of any one of SEQ ID NOs: 4, 6, 8, 10, 12 and 14 has an activity of increasing secretion of a foreign protein.

5. The transformed host cell of claim 1 or 3, wherein said transformed host cell further comprises an isolated polynucleotide selected from the group consisting of (i) and (ii);
(i) an isolated polynucleotide encoding MPD1, SCJ1, ERO1, FKB2, JEM1, LHS1, MPD2, ERJ5, or EUG1 from S. cerevisiae; and
(ii) an isolated polynucleotide encoding ERO1-Lα, ERO1-Lβ, or GRP78 from a human.

6. The transformed host cell of claim 1 or 3, wherein said transformed host cell further comprises a polynucleotide selected from (i) to (vi):
(i) a polynucleotide encoding ERO1 from O. minuta; 
(ii) a polynucleotide encoding ERO1 from a human;
(iii) a polynucleotide encoding Kar2 from O. minuta; 
(iv) a polynucleotide encoding ERO1 from O. minuta, and a polynucleotide encoding Kar2 from O. minuta; 
(v) a polynucleotide encoding ERO1-Lβ from a human, and a polynucleotide encoding GRP78 from a human; and
(vi) a polynucleotide encoding ERO1 from O. minuta, and a polynucleotide encoding GRP78 from a human.

7. The transformed host cell according to claim 1 or 3, wherein the host cell is a eukaryotic cell.

8. The transformed host cell according to claim 7, wherein the eukaryotic cell is a yeast cell.

9. The transformed host cell according to claim 8, wherein the yeast cell is a methanol-assimilating yeast cell.

10. The transformed host cell according to claim 9, wherein the methanol-assimilating yeast cell is Ogataea minuta.

11. The transformed host cell according to claim 8, wherein the yeast cell is Saccharomyces cerevisiae.

12. The transformed host cell of claim 1 or 3, wherein said transformed host cell further comprises a polynucleotide encoding a foreign protein.

13. The transformed host cell according to claim 12, wherein the foreign protein is a multimeric protein.

14. The transformed host cell according to claim 13, wherein the multimeric protein is a heteromultimer.

15. The transformed host cell according to claim 14, wherein the heteromultimer is an antibody or a functional fragment thereof.

16. The transformed host cell according to claim 12, wherein the foreign protein is a glycosyltransferase.

17. A method for producing a foreign protein, comprising culturing the transformed host cell according to claim 12 in a culture medium to express the foreign protein, and isolating the produced foreign protein from the culture medium.

18. The method according to claim 17, wherein the culturing step is conducted under conditions in which protein O-mannosyltransferase (PMT) activity is inhibited.

19. The method according to claim 18, wherein said transferase activity is inhibited by addition of an inhibitor of said transferase activity to the culture medium.

20. The method according to claim 19, wherein said inhibitor is 5-[[3,4-(1-phenylmethoxy)phenyl]methylene]-4-oxo-2-thioxo-3-thiazolidineacetic acid.

21. The method according to claim 18, wherein said transferase activity is inhibited by disrupting said PMT gene.

22. The method according to claim 21, wherein said disruption is disruption of either the PMT5 gene, the PMT6 gene, or both.

23. The method according to claim 18, wherein said transferase activity is inhibited by disrupting said PMT gene and by adding an inhibitor of said transferase activity to the medium.

24. The method according to claim 23, wherein said inhibitor is 5-[[3,4-(1-phenylmethoxy)phenyl]methylene]-4-oxo-2-thioxo-3-thiazolidineacetic acid.

25. An isolated polynucleotide encoding a protein disulfide isomerase (PDI), wherein said isolated polynucleotide is selected from the group consisting of (a) and (b):
(a) an isolated polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1; and
(b) an isolated polynucleotide comprising a nucleotide sequence having at least 95% nucleotide sequence identity to the nucleotide sequence of SEQ ID NO: 1, wherein said nucleotide sequence having at least 95% nucleotide sequence identity to the nucleotide sequence of SEQ ID NO: 1 encodes a protein having an activity of increasing secretion of a foreign protein in a recombinant host cell.

26. An isolated polynucleotide encoding a protein disulfide isomerase (PDI), wherein said isolated polynucleotide encodes a polypeptide selected from the group consisting of (a) and (b):
(a) a polypeptide comprising the amino acid sequence of SEQ ID NO: 2; and
(b) a polypeptide comprising an amino acid sequence having at least 95% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 2, wherein said polypeptide comprising an amino acid sequence having at least 95% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 2 has an activity of increasing secretion of a foreign protein in a recombinant host cell.

27. An expression vector comprising the polynucleotide according to claim 25 or 26.