USOO9068212B2

(12) United States Patent (10) Patent No.: US 9,068,212 B2 Tabuchi et al. (45) Date of Patent: *Jun. 30, 2015

(54) METHOD FOR PRODUCING A 2003. O165495 A1 9, 2003 Carulli et al. POLYPEPTIDE USINGA CELL THAT 2005/0221466 A1 10/2005 Liao et al. 2005/0265983 A1 12/2005 Melamed et al. OVEREXPRESSES A BCARBONATE 2006, OO14937 A1 1/2006 Kang et al. TRANSPORTER 2007/0162995 A1 7/2007 Good et al. 2007, 0166362 A1 7/2007 Sakuma et al. 2007/O190599 A1 8, 2007 Nakano et al. (75) Inventors: Hisahiro Tabuchi, Tokyo (JP); Satoshi 2009,019 1591 A1 7/2009 Tabuchi et al. Tainaka, Tokyo (JP); Tomoya 2009, 0221442 A1 9, 2009 Dower et al. Sugiyama, Tokyo (JP) 2010.0167346 A1 7/2010 Tabuchi et al. 2010/0233759 A1 9/2010 Tabuchi et al. (73) Assignee: Chugai Seiyaku Kabushiki Kaisha, 2010, O248359 A1 9/2010 Nakano et al. 2011/0003334 A1 1/2011 Tabuchi et al. Tokyo (JP) 2012,0045795 A1 2/2012 Tabuchi et al. (*) Notice: Subject to any disclaimer, the term of this patent is extended or adjusted under 35 FOREIGN PATENT DOCUMENTS U.S.C. 154(b) by 0 days. CN 1612689 A 5, 2005 CN 1838969 A 9, 2006 This patent is Subject to a terminal dis EP 1212 619 B1 5/2007 claimer. EP 2213 746 A1 8, 2010 JP 08-191693. A T 1996 (21) Appl. No.: 12/734,283 JP 10-075787 A 3, 1998 JP 10-191984. A 7, 1998 JP 2000-228990 A 8, 2000 (22) PCT Filed: Oct. 23, 2008 JP 2005-525.100 A 8, 2005 JP 2006-506086 A 2, 2006 (86). PCT No.: PCT/UP2008/069184 WO WO92,04381 * 3, 1992 WO WO-97.27485 A1 7/1997 S371 (c)(1), WO WO-01 (20331 A1 3, 2001 (2), (4) Date: Apr. 22, 2010 WO WO-O2/O92768 A2 11/2002 WO WO-03/039485 A2 5, 2003 (87) PCT Pub. No.: WO2009/054433 WO WO-2005, O76O15 A1 8, 2005 WO WO-2006/006693 A1 1, 2006 PCT Pub. Date: Apr. 30, 2009 WO WO-2006, 119115 A2 11/2006 WO WO-2007/056507 A1 5/2007 (65) Prior Publication Data WO WO-2007,1 19774 A1 10, 2007 WO WO-2008, 114673 A1 9, 2008 US 2011/0014654 A1 Jan. 20, 2011 WO WO-2009/02O144 A1 2, 2009 WO WO-2009/051109 A1 4/2009 (30) Foreign Application Priority Data WO WO-2009/054433 A1 4/2009 OTHER PUBLICATIONS Oct. 24, 2007 (JP) ...... 2007-276182 Yang et al., J. Biol. Chem. 281:34.525-34536, 2006.* (51) Int. Cl. Wu et al., Chinese J. Physiol. 49:192-198, 2006.* CI2P 21/02 (2006.01) Lux et al., Proc. Natl. Acad. Sci. USA 86:9089-9093, 1989.* CI2N5/00 (2006.01) GenBank Accession No. EGWO 1898, Aug. 2011, 2 pages.* CI2N 5/07 (2010.01) GenBank Accession No. AEQ38544, Oct. 2011, 2 pages.* CI2N 5/16 (2006.01) Kondo et al., Oncogene 17:2585-2591, 1998.* C07K 6/28 (2006.01) Shen et al., Neoplasia 9:812-819, Oct. 2007.* C07K 6/30 (2006.01) Romero et al., Mol. Aspects Med. 34:159-182, 2013.* Chambard et al., J. Physiol. 550.3:667-677, 2003.* (52) U.S. Cl. International Search Report mailed Nov. 25, 2008 in PCT/JP2008/ CPC ...... CI2P21/02 (2013.01); C07K 16/2866 0.69184, 5 pages. (2013.01); C07K 16/303 (2013.01); C07K Alper, Seth L., “Molecular physiology of SLC4 anion exchangers.” 2317/14 (2013.01); C12N 2510/02 (2013.01) Exp. Physiol., 2006, 91:153-161. (58) Field of Classification Search None (Continued) See application file for complete search history. Primary Examiner — David J Steadman (56) References Cited (74) Attorney, Agent, or Firm — Foley & Lardner LLP U.S. PATENT DOCUMENTS (57) ABSTRACT 5,658,786 A 8, 1997 Smith et al. The present invention provides a method capable of produc 6,184,007 B1 2/2001 Dusch et al. ing a protein efficiently. A method of producing a polypep 6,225,115 B1 5, 2001 Smith et al. tide, comprising culturing a cell which strongly expresses a 6,251,613 B1 6/2001 Kishimoto et al. bicarbonate transporter and has a transferred DNA encoding 6,316,238 B1 1 1/2001 Nakamura et al. 6,812,339 B1 1 1/2004 Venter et al. a desired polypeptide and thereby allowing the cell to produce 7,413,536 B1 8, 2008 Dower et al. said polypeptide. 7.919,086 B2 4/2011 Nakano et al. 8,741,601 B2 * 6/2014 Tabuchi et al...... 435/69.6 7 Claims, 10 Drawing Sheets US 9,068.212 B2 Page 2

(56) References Cited decarboxylase.” XP002597738 retrieved from EBI accession No. UNIPROT:Q9DBEO Database accession No. Q9DBEO, 2 pages. OTHER PUBLICATIONS Database Uniprot Online Mar. 15, 2005, XP002593030, retrieved Database DDBJ/EMBL/GenBank online. Accession No. from EBI accession No. UNIPROT:Q5F431, 1 page. NM 000342, uploaded Sep. 25, 2007, Keskanokwong et al., Defi Database Uniprot Online Oct. 1, 2000, XP002593031, retrieved nition: Homo sapiens solute carrier family 4, anion exchanger, mem from EBI accession No. UNIPROT:Q9MZ34, 2 pages. ber 1 (erythrocyte membrane protein band 3. Diego blood group) de la Cruz Edmonds et al., “Development of Transfection and High (SLC4A1), mRNA, retrieved Nov. 11, 2008, 12 pages. 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No. osmolyte in preimplantation mouse embryos cultured from fertilized 13/368,945. eggs.” Molecular Reproduction and Development, Oct. 2003, Tanner et al., “The complete amino acid sequence of the human 66(2): 153-161. erythrocyte membrane anion-transport protein deduced from the Han et al., “Is TauT an Anti-Apoptotic Gene? Taurine 6, Oja et al. cDNA sequence.” Biochem. J., 1988, 256:703-712. Eds., 2006, 59-67. Final Office Action dated Dec. 17, 2010 in U.S. Appl. No. Hwang et al., “Expression and purification of recombinant human 12/226,195. angiopoietin-2 produced in Chinese hamster ovary cells.” Protein Final Office Action dated Aug. 23, 2011 in U.S. Appl. No. Expression and Purification, 2005, 39: 175-183. 12/733,815. Ifandiet al., “Regulation of Cell Proliferation and Apoptosis in CHO Notice of Allowance dated Dec. 20, 2012, in U.S. Appl. No. K1 Cells by the Coexpression of c-Myc and Bcl-2.” Biotechnol. 12/733,052. Prog., 2005, 21:671-677. 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Amplification and Their Stability in the Absence of Selective Pres Christensen et al., “High expression of the taurine transporter TauTin sure.” Biotechnology and Bioengineering, Apr. 5, 1998, 58(1):73-84. primary cilic of NIH3T3 fibroblasts.” Cell Biology International, Lee et al., “Development of Apoptosis-Resistant Dihydrofolate 2005, 29:347-351. Reductase-Deficient Chinese Hamster Ovary Cell Line.” Biotechnol. Christie et al., “The Adaptation of BHK Cells to a Non-Am Bioengineer, 2003, 82:872-876. moniagenic Glutamate-Based Culture Medium. Biotechnology and Liu et al., "Cloning and expression of a cDNA encoding the trans Bioengineering, Aug. 5, 1999, 64(3):298-309. porter oftaurine and B-alanine in mouse brain.” Proc. Natl. Acad. Sci. Database EMBL Online Jul. 23, 1992, XP002593029, retrieved USA, Dec. 1992, 89(24): 12145-12149. from EBI accession No. EMBL: M95495, 3 pages. Miyasaka et al., "Characterization of Human Taurine Transporter Database Uniprot Online Jan. 10, 2006, XP002593032, retrieved Expressed in Insect Cells Using a Recombinant Baculovirus.” Pro from EBI accession No. UNIPROT:Q2VRP7, 1 page. tein Expression and Purification, 2001, 23(3):389-397. Database UniProt Online Jul. 1, 1993, XP002593028, retrieved Ngo et al., “Computational Complexity, Protein Structure Prediction, from EBI accession No. UNIPROT:Q00589, 2 pages. and the Levinthal Paradox.” The Protein Folding Problem and Ter Database UniProt Online Jun. 1, 2001, “RecName: Full=Cysteine tiary Structure Prediction, Merz et al. (Eds.), 1994,433 and 492-495. sulfinic acid decarboxylase; EC=4.1.1.29; AltName: Full=Cysteine Office Action dated May 12, 2011 in U.S. Appl. No. 12/733,052. Sulfinate decarboxylase; AltName: Full=Sulfinoalanine Office Action dated Aug. 9, 2011 in U.S. Appl. No. 12/450,161. US 9,068.212 B2 Page 3

(56) References Cited Tinland et al., “Agrobacterium tumefaciens transfers single-stranded transferred DNA (T-DNA) into the plant cell nucleus.” Proc. Natl. OTHER PUBLICATIONS Acad. Sci. USA, Aug. 1994, 91:8000-8004. Trill et al., “Production of monoclonal antibodies in COS and CHO Porter et al., “Non-steady-state kinetics of brain glutamate cells.” Current Opinion in Biotechnology, 1995, 6:553-560. decarboxylase resulting from interconversion of the apo- and Uchida et al., “Molecular cloning of the cDNA for an MDCK cell holoenzyme.” Biochimica et Biophysica Acta, 1988,874.235-244. Na+- and Cl—dependent taurine transporter that is regulated by Ramamoorthy et al., “Functional characterization and chromosomal hypertonicity.” Proc. Natl. Acad. Sci. USA, Sep. 1992, 89:8230 localization of a cloned taurine transporter from human placenta.” 8234. Biochem. J., 1994, 300:893-900. Voss et al., “Regulation of the expression and Subcellular localization Reymond et al., “Molecular cloning and sequence analysis of the of the taurine transporter TauT in mouse NIH3T3 fibroblasts.” Eur, J. cDNA encoding rat liver cysteine sulfinate decarboxylase (CSD).” Biochem., 2004, 271:4646-4658. Biochimica et Biophysica Acta, 1996, 1307:152-156. Wirth et al., “Isolation of overproducing recombinant mammalian Rudinger, J., “Characteristics of the amino acids as components of a cell lines by a fast and simple selection procedure.” Gene, 1988, peptide hormone sequence.” Peptide Hormones, Parsons (Ed.), 1976, 73:419-426. 1-7. Yang et al., “cDNA Cloning, Genomic Structure, Chromosomal Smith et al., "Cloning and Expression of a High Affinity Taurine Mapping, and Functional Expression of a Novel Human Alanine Transporter from Rat Brain.” Mol. Pharmacol., 1992,42(4):563-569. Aminotransferase.” Genomics, Mar. 1, 2002, 79(3):445-450. Tabuchi et al., “Overexpression of Taurine Transporter in Chinese Zhang et al., “Metabolic characteristics of recombinant Chinese Hamster Ovary cells Can Enhance Cell Viability and Product Yield, hamster ovary cells expressing glutamine synthetase in presence and While Promoting Glutamine Consumption.” Biotechnology and absence of glutamine.” Cytotechnology, 2006, 51(1):21-28. Bioengineering, 2010, 107(6):998-1003. Han et al., “Mechanisms of regulation of taurine transporter activity.” Tang et al., “Protein Phosphorylation and Taurine Biosynthesis. In Taurine 6, Edited by Oja and Saransaari, 2006, 79-90. Vivo and In Vitro.” Journal of Neuroscience, Sep. 15, 1997. Herman et al., "Low dose methotrexate induces apoptosis with reac 17(18):6947-6951. tive oxygen species involvement in T lymphocytic cell lines to a Tappaz et al., “Characterization of the cDNA Coding for Rat Brain greater extent than in monocytic lines.” Inflammation Research, Cysteine Sulfinate Decarboxylase: Brain and Liver Enzymes are 2005, 54:273-280. Identical Proteins Encoded by Two Distince mRNAs.” J. Neurochem., 1999, 73(3):903-912. * cited by examiner U.S. Patent Jun. 30, 2015 Sheet 1 of 10 US 9,068,212 B2

U.S. Patent Jun. 30, 2015 Sheet 2 of 10 US 9,068,212 B2

Fig. 2

SWa 7795 Sgr A7801

hygromycin resistance ORF EcoR 6303

EcoRV 649

Sac 222 Sf Promote Barn 5845 Not 221B SV40 polyA Barn 2238 mCMTV promoter f enh

EcoR 2862

Barn 341 EcoRW 424 U.S. Patent Jun. 30, 2015 Sheet 3 of 10 US 9,068,212 B2

Fig. 3

Pvt 862 Ne 813 Hind 340 Sal 540 puromycin resistance ORF

Py 6375 Bar 1391 Sca. 6264 CMW promoter f enh

EcoR 5749 EcoRI 2015 SWall 5732

BamH2S70

Bar A998 SV40 polyA Eco R 3177

Fig. 4

1200 1200 Antibody Yield

1000 OOO pHyg-AE1 (n = 4) ( 927. 46) mg/L. 800 800 pHyg (n = 4) (786-115) mg/L. t Test PCO.05 600 600 pHyg-AE1 pHyg U.S. Patent Jun. 30, 2015 Sheet 4 of 10 US 9,068,212 B2

Fig. 5

1500 1500 O Antibody Yield

1200 1200 O AE1/CSAD (n = 9) (1098 it 139) mg/L. O 900 AE1/ppurf (n =r 8) 900 ( 975 + 122) mg/L. t Test PacO.05 600 600 AE1/CSAD AE1/ppur

Fig. 6

80 80 O Survival Ratio ae 70 70 s AE1/CSAD (n = 9) O O (69 + 4) % 60 60 r AE1/pPur (n = 8) S (56 - 4) % 50 50 (7. t Test P-O.O1 40 40 AE1/CSAD AE1/ppur U.S. Patent Jun. 30, 2015 Sheet 5 of 10 US 9,068,212 B2

Fig. 7

S 1600 1600 g Antibody Yield e 3 1200 1200 AE1/ALT1 (n = 10) (1099 E211) mg/L. CYO

ls O AE1/pFur (n = 8) 800 800 ( 766 - 73) mg/L. 3. O t Test PCO.01 CC 400 400 AE1/ALT1 AE1/pPur

Fig. 8 AE1/ALT-Coexpressing Strain AA53 O Yield On Day 7 of Culture 1.9 g/L TauT/ALT-Coexpressing Strain TA41 Yield On Day 7 of Culture 1.5 g/L

O 7 14 Day of Culture U.S. Patent Jun. 30, 2015 Sheet 6 of 10 US 9,068.212 B2

888 8388 Sa: ; 38 3888ty ci: 83888 88&:

U.S. Patent Jun. 30, 2015 Sheet 7 of 10 US 9,068,212 B2

Fig. 11

Pyu 662) Nde 6571 hind 340 Sai SO puronycin resistance ORF

Banh. 1391 Pvt 133 pPur-ALT1 Sca. 522 6622 bp CMV

promoter f enh

EcoR 47 human ALT1 EcoR 2015

SWall 4490 SV40 polyA

Ban 3756 Sal 3519

US 9,068,212 B2 1. 2 METHOD FOR PRODUCINGA Non-Patent Document 1 POLYPEPTIDE USINGA CELL THAT van Adelsberg J S. et al., J Biol Chem 1993; 268:11283 OVEREXPRESSES A BICARBONATE 11289 TRANSPORTER Non-Patent Document 2 Shayakui C. et. al., Curr Opin Nephrol Hypertens 2000; CROSS-REFERENCE TO RELATED 9:541-546 APPLICATIONS Non-Patent Document 3 AlperS L. et al., Annu Rev Physiol 2002: 64:899-923 This application is a National Stage application of PCT/ Non-Patent Document 4 JP2008/069184, filed Oct. 23, 2008, which claims priority 10 Komlosi P. et al., Am J Physiol Renal Physiol 2005; 288: from Japanese application JP 2007-276182, filed Oct. 24. F38O-F386 2007. Non-Patent Document 5 Gawenis L. R. et. al., J Biol Chem 2004; 279:30531-30539 TECHNICAL FIELD Non-Patent Document 6 15 Melvin et al, J Biol Chem 1999; 274:22855-22861 The present invention relates to a cell to be used in the Non-Patent Document 7 production of heteroproteins and a production method using Koet al., EMBO J. 2002; 21:5662-5672 the cell. In more detail, the present invention relates to a cell Non-Patent Document 8 that strongly expresses a bicarbonate transporter and a Soleimani et al., Am. J. Physiol. Renal Physiol. 2001: 280: method for producing a polypeptide using the cell. 2O F356-F364 Non-Patent Document 9 BACKGROUND ART Wang et al., Am. J. Physiol. Gastrointest. Liver Physiol. 2002: 282:G573-G579 When proteins useful as pharmaceuticals are produced Non-Patent Document 10 with the recombinant DNA technique, use of animal cells 25 Petrovic et al., Am. J. Physiol. Renal Physiol. 2004; 286: enables complicated post-translational modification and F161-F169 folding which prokaryotic cells can not perform. Therefore, Non-Patent Document 11 animal cells are frequently used as host cells for producing Xu et al., Am. J. Physiol. Cell Physiol. 2005; 289:C493-C505 recombinant proteins. Recently, a large number of biopharmaceuticals, such as 30 DISCLOSURE OF THE INVENTION antibodies and physiologically active proteins, have been developed. Techniques that permit efficient production of Problem for Solution by the Invention recombinant proteins by animal cells lead to cost reduction of biopharmaceuticals and promise their stable Supply to It is an object of the present invention to provide a method patients. 35 which is capable of producing a polypeptide efficiently. Under these circumstances, a method of protein production with higher production efficiency is desired. Means to Solve the Problem An anion exchanger is a transporter that mediates antiport of intracellular and extracellular anions across a plasma As a result of extensive and intensive researches toward the membrane (membrane transport protein). An SLC4 family is 40 solution of the above problem, the present inventors have a family of HCO transporters, and three members belong found that it is possible to increase the yield of a desired ing to the SLC4 family, namely AE1, AE2, and AE3, have a polypeptide by using a cell that strongly expresses a bicar function to exchange Cl outside a plasma membrane for bonate transporter. Thus, the present invention has been HCO inside a plasma membrane. achieved. Moreover, the desired polypeptide could be pro In a kidney, AE1 is found in a intercalated cells in collect- 45 duced in an even greater amount by using cells capable of ing ducts in the basolateral membrane (Non-Patent Docu co-expressing a bicarbonate transporter and cysteine Sulfinic ment 1). It has been known that mutations in human AE1 acid decarboxylase (hereinafter sometimes referred to as cause distal renal tubular acidosis (Non-Patent Documents 2 “CSAD') or alanine aminotransferase (hereinafter some and 3). times referred to as "ALT"). Further, in a kidney, three isoforms of AE2, namely AE2a, 50 The present invention may be summarized as follows. AE2b, and AE2c, have been found. AE2 is considered to (1) A method of producing a polypeptide, comprising cultur regulate intracellular pH homeostasis for cell signal transduc ing a cell which strongly expresses a bicarbonate transporter tion (Non-Patent Document 4). However, an AE2 knockout and has a transferred DNA encoding a desired polypeptide mouse that dies during the weaning period has been found to and thereby allowing the cell to produce said polypeptide. suffer no renal phenotypic abnormalities (Non-Patent Docu- 55 (2) The method of (1) above, wherein the cell which strongly ment 5). expresses a bicarbonate transporter is a cell into which a DNA An SLC26 is a relatively new anion exchanger family, and encoding the bicarbonate transporter has been transferred. it has been Suggested that a large number of its members (for (3) The production method of (1) or (2) above, wherein the example, SLC26A3, SLC26A4, SLC26A6, and SLC26A9) cell that strongly expresses a bicarbonate transporter further are bicarbonate exchangers (Non-Patent Documents 6 to 11). 60 expresses cysteine Sulfinic acid decarboxylase or alanine On the other hand, it has been absolutely unknown that by aminotransferase strongly. strongly expressing an anion exchanger having a bicarbonate (4) The production method of any one of (1)–(3) above, transporter function, uptake of anions into a cultured cell and wherein the bicarbonate transporter is an SLC4 anion excretion of anions to the outside of the cell, as mediated by exchanger or SLC26 anion exchanger. the anion exchanger, can be artificially promoted, which con- 65 (5) The production method of any one of (1)–(3) above, tributes to improvement in the production of a desired recom wherein the bicarbonate transporter is an SLC4 anion binant protein in the cultured cell. exchanger. US 9,068,212 B2 3 4 (6) The production method of (5) above, wherein the SLC4 FIG. 5 is a plot of the amount of anti-glypican-3 antibody anion exchanger is AE1. production on day 10 offed-batch culture in a 50-mL shaker (7) The method of any one of (1)–(6) above, wherein the cell flask. The amount of an anti-glypican-3 antibody produced by is Chinese hamster ovary cells. an AE1/CSAD co-expressing cell strain (n=9) which was (8) The method of any one of (1)-(7) above, wherein the obtained by introducing pPur-CSAD into a pHyg-AE1-42 desired polypeptide is an antibody. strain, or a pHyg-AE1-transformed cell capable of high-yield (9) The method of any one of (4)–(6) above, wherein the DNA antibody production, was significantly greater than that pro encoding the SLC4 anion exchanger is any one of the follow duced by AE1/ppur co-expressing cells (n=8) which were ing (a) to (e): obtained by introducing pPur into a pHyg-AE1-42 strain (a) a DNA encoding a polypeptide having the amino acid 10 (P<0.05). sequence as shown in SEQID NO: 2; FIG. 6 is a plot of survival rates on day 10 of fed-batch (b) a DNA encoding a polypeptide which has an amino acid culture in a 50-mL shaker flask. The survival rate of an AE1/ sequence derived from the amino acid sequence as shown in CSAD co-expressing cell strain (n=9) which was obtained by SEQID NO:2 by substitution, deletion, addition and/or inser 15 introducing pPur-CSAD into a pHyg-AE1-42 strain, or a tion of one or more amino acid residues and yet has SLC4 pHyg-AE1-transformed cell capable of high-yield antibody anion exchanger activity; production, was significantly higher than that of AE1/ppur (c) a DNA encoding a polypeptide having 50% or more co-expressing cells (n=8) which were obtained by introduc amino acid sequence homology with the amino acid sequence ing pPur into a pHyg-AE1-42 strain (P<0.01). as shown in SEQ ID NO: 2 and yet having SLC4 anion The survival rates on day 7 of the culture were also char exchanger activity; acterized by P-0.01 (data not shown). (d) a DNA having the nucleotide sequence as shown in FIG. 7 is a plot of the amount of anti-glypican-3 antibody SEQID NO: 1; production on day of 8 fed-batch culture in a 50-mL shaker (e) a DNA which hybridizes to a DNA complementary to a flask. The amount of an anti-glypican-3 antibody produced by DNA having the nucleotide sequence as shown in SEQ ID 25 an AE1/ALT co-expressing cell strain (n=10) which was NO: 1 under stringent conditions and yet encodes a polypep obtained by introducing pPur-ALT1 into a pHyg-AE1-42 tide having SLC4 anion exchanger activity. strain, or a pHyg-AE1-transformed cell capable of high-yield (10) A method of preparing a pharmaceutical containing a antibody production, was greater than that produced by an polypeptide prepared by the method of any one of (1)–(9) AE1/CSAD strain (n=9), and further, the amount of the anti above. 30 glypican-3 antibody produced by an AE1/ALT co-expressing (11) A cell which has a transferred DNA encoding a bicar cell strain was significantly greater than that produced by bonate transporter and a transferred DNA encoding a desired AE1/pPur co-expressing cells (n=8) which were obtained by polypeptide. introducing pPur into a pHyg-AE1-42 strain (P<0.01). (12) The cell according to (11) above, which further has a FIG. 8 is a graph showing the amount of an antibody transferred DNA encoding cysteine sulfinic acid decarboxy 35 lase or alanine aminotransferase. produced by AA53, or an AE1/ALT1 co-expressing strain, (13) A cell which has a transferred DNA encoding a bicar during fed-batch culture in a 1 L-ar. The amount of anti bonate transporter and a transferred DNA encoding cysteine glypican-3 antibody production on day 7 of the culture was Sulfinic acid decarboxylase or alanine aminotransferase. 1.9 g/L. 40 FIG.9 shows the nucleotide sequence (SEQ ID NO:3) of Effect of the Invention a newly cloned, CHO cell-derived hamster CSAD gene and the amino acid sequence (SEQID NO: 4) deduced therefrom. According to the present invention, it has become possible FIG. 10 shows a plasmid for Puromycin selection which to produce a desired polypeptide in high yield. was used for expressing hamster CSAD (493 amino acids). The present specification encompasses the contents dis 45 FIG. 11 shows a plasmid for Puromycin selection which closed in the specification and/or the drawings of Japanese was used for expressing human ALT1 (496 amino acids). Patent Application No. 2007-276182 based on which the FIG. 12 is a graph showing the amount of an antibody present patent application claims priority. produced by an anti IL-6R antibody producing AE1-S08 cell derived from an AE1 strongly expressing host during fed BRIEF DESCRIPTION OF THE DRAWINGS 50 batch culture in a 1 L-jar. The amount of anti-IL-6R antibody production on day 14 of the culture was 3.0 g/L. FIG. 1 shows an AE1 membrane topology produced based on a transmembrane domain and direction predicted from an FIG. 13 shows the nucleotide sequence (SEQID NO: 7) of amino acid sequence of human hepatic cell-derived AE1 as a newly cloned, CHO cell-derivedhamster taurine transporter obtained by TMpred program with reference to FIG. 1 in Exo 55 gene and the amino acid sequence (SEQID NO: 8) deduced Physiol 91.1 pp. 153-161, 2006, Seth L. Alper (SEQID NO: therefrom. 11). FIG. 14 is a taurine transporter membrane topology which FIG. 2 shows a plasmid for Hygromycin-selection, in was created based on the transmembrane regions and orien which human AE1 (911 amino acids) has been expressed. tations predicted by TMpred program from the amino acid FIG.3 shows a plasmid for Puromycin-selection, in which 60 sequence of a newly cloned, CHO cell-derived hamster TauT human AE1 (911 amino acids) has been expressed. with reference to FIG. 5 of Shinichi Uchida et al., Proc. Natl. FIG. 4 is a plot of the amount of anti-glypican-3 antibody Acad. Sci. USA Vol. 89, pp. 8230-8234, September 1992. production on day 12 offed-batch culture in a 50-mL shaker Mark (9 indicates hamster TauT specific amino acid residues. flask. The amount of an anti-glypican-3 antibody produced by A large number of amino acid residues different from those in pHyg-AE1-transformed cells (n=4) was significantly greater 65 human TauT are present in the 2nd loop (EX: extra-cell mem than that produced by pHyg-transformed cells (n=4) brane region), the 12th transmembrane region (TM) and the (P<0.05). C-terminal (IC: intracellular region). US 9,068,212 B2 5 6 FIG. 15 shows a plasmid for Hygromycin-selection, which The SLC4 anion exchanger may be exemplified by SLC4A1 was used for expressing hamster TauT (622 amino acids). (AE1), SLC4A2 (AE2), SLC4A3 (AE3), SLC4A4 (NBCel), SLC4A5 (NBCe2), SLC4A7 (NBCn1), SLC4A8 (kNBC3), BEST MODE FOR CARRYING OUT THE SLC4A9 (NBCn2), SLC4A10 (NBCn3), and SLC4A11 INVENTION (NaBC1), among which AE1 is preferable. An SLC26 anion exchanger is a multifunctional membrane Hereinbelow, embodiments of the present invention will be protein that acts in almost all organ systems. For the SLC26 described in more detail. anion exchanger, one that mediates antiport of sulfate anions, The present invention provides a method of producing a iodide anions, formate anions, oxalate anions, chloride polypeptide, comprising culturing a cell which strongly 10 anions, hydroxyl anions, bicarbonate anions and the like, and expresses a bicarbonate transporter and has a transferred a chloride ion channel, or an anion-dependent molecular DNA encoding a desired polypeptide and thereby allowing motor exist. The SLC26 anion exchanger is considered to be the cell to produce the polypeptide. involved in homeostasis of various anions and 10 kinds In the method of the present invention, the cell may be (SLC26A1, SLC26A2, SLC26A3, SLC26A4, SLC26A5, eithera natural cell capable of producing the desired polypep 15 SLC26A6, SLC26A7, SLC26A8, SLC26A9, and tide or a transformed cell into which a DNA encoding the SLC26A11) of anion exchanger families have been known. desired polypeptide has been transferred. Preferably, a trans For example, SLC26A3, SLC26A4, SLC26A6 and formed cell into which a DNA encoding the desired polypep SLC26A9, which are transporters for hydroxyl anions and tide has been transferred is used. bicarbonate anions regulate pH inside as well as outside a In the method of the present invention, the desired polypep membrane in a similar manner to an SLC4 anion exchanger. tide is not particularly limited. The polypeptide may be any SLC26A1 SLC26A2, SLC26A4, SLC26A6, SLC26A9 and polypeptide such as an antibody (e.g., anti-IL-6 receptor anti SLC26A11 are expressed in a kidney. SLC26A1 transports body, anti-glypican-3 antibody, anti-CD3 antibody, anti sulfate anions and oxalate anions whereas SLC26A6 medi CD20 antibody, anti-GPIb/IIIa antibody, anti-TNF antibody, ates antiport of various anions in order to take up sodium anti-CD25 antibody, anti-EGFR antibody, anti-Her2/neu 25 chloride. SLC26A1, SLC26A4 and SLC26A6 and SLC26A5 antibody, anti-RSV antibody, anti-CD33 antibody, anti become causative factors for nephrolithiasis, hypertension, CD52 antibody, anti-IgE antibody, anti-CD11a antibody, and hearing loss, respectively. SLC26A7 is involved in acid anti-VEGF antibody, anti-VLA4 antibody, and the like) or a base homeostasis and blood pressure control in a similar physiologically active protein (e.g., granulocyte-colony manner to SLC26A4. The SLC26 anion exchanger may be stimulating factor (G-CSF), granulocyte macrophage-colony 30 exemplified by SLC26A1, SLC26A2, SLC26A3, SLC26A4, stimulating factor (GM-CSF), erythropoietin, interferon, SLC26A5, SLC26A6, SLC26A7, SLC26A8, SLC26A9, and interleukin such as IL-1 or IL-6, t-PA, urokinase, serum albu SLC26A11. min, blood coagulation factor, PTH, and the like). An anti A cell which strongly expresses a bicarbonate transporter body is particularly preferred, and may be any antibody Such is not particularly limited as long as the cell has an increased as a natural antibody, a low molecular sized antibody (e.g., 35 expression level of a bicarbonate transporter compared to a Fab, scFv, sc(FV)2), a chimeric antibody, a humanized anti corresponding natural cell. The natural cell is not particularly body, etc. limited. A cell which is used as a host in the production of a By using strongly a bicarbonate transporter expressing recombinant protein (e.g., CHO cells) may be used. cells, the amount of a polypeptide produced by cells can be A bicarbonate transporter to be strongly expressed in a cell increased. 40 may be derived from any organism and no particular limita A bicarbonate transporter is a membrane protein that has tion is imposed thereon. Specifically, the bicarbonate trans an antiport function, by which bicarbonate anions (HCO) or porter may be derived from organisms including a human, carbonate anions (CO) are excreted whereas chloride rodents such as a mouse, a rat, and a hamster, mammals such anions and Sulfate anions are taken up. A bicarbonate trans as a chimpanzee, a cow, a horse, a dog, and a wolf, birds such porter may be exemplified by an SLC4 anion exchanger and 45 as a chicken, fishes such as a Zebrafish and an eel, and insects an SLC26 anion exchanger. such as Drosophila; the bicarbonate transporter is preferably An SLC4 anion exchanger is a membrane protein that derived from a human, rodents, or the same species as the host regulates intracellular pH homeostasis and cell Volume. At cell. For example, in a case where the cell in which a bicar present, 10 kinds (SLC4A1 (AE1), SLC4A2 (AE2), SLC4A3 bonate transporter is to be strongly expressed is a Chinese (AE3), SLC4A4 (NBCe1), SLC4A5 (NBCe2), SLC4A7 50 hamster ovary cell (CHO cell), the bicarbonate transporter is (NBCn1), SLC4A8(kNBC3), SLC4A9 (NBCn2), SLC4A10 preferably derived from a human or a hamster. (NBCn3), and SLC4A11 (NaBC1)) of SLC4 families are The cell which strongly expresses a bicarbonate trans known, and at least one kind of isoform exists. These SLC4 porter may be any cell, for example, eukaryotic cell Such as anion exchangers have different functions; for example, animal, plant and yeast cells, prokaryotic cell Such as E. coli SLC4A1 (AE1), SLC4A2 (AE2), ALC4A3 (AE3), and 55 and B. subtilis, etc. Preferably, animal cells such as CHO and ALC4A9 (NBCn2 or AE4) are non-Na'-dependent, electri COS cells are used, CHO cells are particularly preferred. In cally-neutral exchangers for Cl and HCO, ALC4A4 order to prepare a desired polypeptide, cells Suitable for trans (NBCe1) and ALC4A5 (NBCe2) are electrogenic, ALC4A7 fer of a gene encoding the desired polypeptide Such as CHO (NBCn1) is an electrically-neutral cotransporter for Na" and dhfr-cells are preferred. HCO, ALC4A8 (kNBC3) and ALC4A10 (NBCn3) are 60 As a cell which strongly expresses a bicarbonate trans Na'-dependent, electrically-neutral exchangers for Cl and porter, a cell into which a bicarbonate transporter gene (e.g., HCO and ALC4A11 (NaBC1) is an electrogenic cotrans SLC4 anion exchanger gene, SLC26 anion exchanger gene, porter for Na" and borate. The above SLC4 anion exchangers etc.) has been artificially transferred may be given. A cell into have a site-specific action. For example, in a case of AE1, AE1 which a bicarbonate transporter gene has been artificially present in polar epithelial cells contributes to transepithelial 65 transferred can be prepared by methods known to those secretion and resorption of acids and bases whereas AE1 skilled in the art. For example, such a cell may be prepared by present in erythrocytes of trout promotes osmolyte transport. incorporating a bicarbonate transporter into a vector and US 9,068,212 B2 7 8 transforming the vector into a cell. Furthermore, the concept activity is functionally equivalent to an SLC4 anion of “cells into which a bicarbonate transporter gene has been exchanger derived from human, mouse, rat, chimpanzee, artificially transferred encompasses herein cells in which an cow, horse, dog, wolf, chicken or Zebrafish (hereinafter some endogenous bicarbonate transporter gene has been activated times referred to as “SLC4 anion exchanger derived from by gene activation technology (see, for example, Interna human or the like'). Such a polypeptide encompasses, for tional Publication WO94/12650) so that the bicarbonate example, mutants of the SLC4 anion exchanger derived from transporter is strongly expressed. human or the like. In Example described below, a mutant in As an SLC4 anion exchanger gene to be transferred in a which four out of 911 amino acids were replaced (L88R, cell, any one of the following DNAS (a) to (e) encoding an E693G, V732A and H834Y) was used. SLC4 anion exchanger may be used. 10 As methods well-known to those skilled in the art for (a) a DNA encoding a polypeptide having the amino acid preparing polypeptides functionally equivalent to a specific sequence as shown in SEQID NO: 2; polypeptide, methods of introducing mutations into polypep (b) a DNA encoding a polypeptide which has an amino acid tides may be given. For example, those skilled in the art could sequence derived from the amino acid sequence as shown in prepare polypeptides functionally equivalent to the SLC4 SEQID NO:2 by substitution, deletion, addition and/or inser 15 tion of one or more amino acid residues and yet has SLC4 anion exchanger derived from human or the like by appropri anion exchanger activity; ately introducing mutations into amino acids of the SLC4 (c) a DNA encoding a polypeptide having 50% or more anion exchanger derived from human or the like by site amino acid sequence homology with the amino acid sequence directed mutagenesis (Hashimoto-Gotoh, T. et al. (1995) as shown in SEQ ID NO: 2 and yet having SLC4 anion Gene 152,271-275: Zoller, MJ, and Smith, M. (1983) Meth exchanger activity; ods Enzymol. 100, 468-500; Kramer, W. et al. (1984) Nucleic (d) a DNA having the nucleotide sequence as shown in Acids Res. 12,9441-9456; Kramer W, and Fritz HJ (1987) SEQID NO: 1; Methods. Enzymol. 154, 350-367; Kunkel, TA (1985) Proc (e) a DNA which hybridizes to a DNA complementary to a Natl Acad Sci USA. 82, 488-492: Kunkel (1988) Methods DNA having the nucleotide sequence as shown in SEQ ID 25 Enzymol. 85,2763-2766). Mutations in amino acids may also NO: 1 under stringent conditions and yet encodes a polypep occur in nature. tide having SLC4 anion exchanger activity. Specific examples of polypeptides functionally equivalent The concept of an SLC4 anion exchanger activity encom to the SLC4 anion exchanger derived from human or the like passes an activity to take up C1 and SO, present in the include, but are not limited to, a polypeptide having an amino medium and excrete intracellular HCO, and borate in order 30 acid sequence derived from the amino acid sequence (e.g., to maintain intracellular pH homeostasis and cell Volume. SEQID NOS: 2) of the SLC4 anion exchanger derived from The SLC4 anion exchanger activity can be measured as human or the like by deletion of one or more amino acids, follows. preferably 1-30 amino acids, more preferably 1-10 amino Cells in which SLC4 is functionally expressed are treated acids; a polypeptide having an amino acid sequence derived with BCECF-AM which is a pH-sensitive dye. Then, fluores 35 from the amino acid sequence of the SLC4 anion exchanger cent intensity is compared between cells that have been per derived from human or the like by addition of one or more fused with a medium containing CF and Na" and cells that amino acids, preferably 1-30 amino acids, more preferably have been perfused with a medium free of Cl and Na", 1-10 amino acids; and a polypeptide having an amino acid whereby changes in intracellular pH (pHi) can be measured sequence derived from the amino acid sequence of the SLC4 (Dahl N K. et al., J Biol Chem 2003: 278:44949-44958; 40 anion exchanger derived from human or the like by Substitu Fujinaga J. et al., J Biol Chem 1999; 274:6626-6633). tion of one or more amino acids, preferably 1-30 amino acids, In the present invention, a DNA encoding a polypeptide more preferably 1-10 amino acids, with other amino acids. having the amino acid sequence of SEQID NO: 2 is advan Amino acid residues to be mutated are not particularly tageously used as a DNA encoding an SLC4 anion exchanger. limited. Preferably, amino acid residues are mutated to other Besides that, a DNA encoding a polypeptide having the 45 amino acids in which the nature of the initial amino acid side amino acid sequence of SEQ ID NO: 2 in which one or a chain is conserved. Specific examples of the nature of amino plurality (for example, several) of amino acid(s) is/are Sub acid side chain include hydrophobic amino acids (A, I, L., M. stituted, deleted, added, and/or inserted, and also having an F. P. W. Yand V), hydrophilic amino acids (R, D., N, C, E, Q, SLC4 anion exchanger activity may be used. The amino acid C, H, K, Sand T), amino acids with an aliphatic side chain (G: sequence of SEQ ID NO: 2 is an amino acid sequence of 50 A. V. L. I and P), amino acids with a hydroxyl group-contain human AE1. Aside from the sequence information of human ing side chain (S. T and Y), amino acids with a Sulfur atom AE1, the counterpart information about a mouse, a rat, a containing side chain (C and M), amino acids with a carboxy chimpanzee, a cow, a horse, a dog, a wolf, a chicken, a lic acid and amide-containing side chain (D, N., E and Q), Zebrafish, and the like has been registered as mouse; Gen amino acids with a base-containing side chain (R, K and H) Bank NM 011403, rat; GeneBank NM 012651, chimpan 55 and amino acids with an aromatic-containing side chain (H. F. Zee: GenBank XM 001151353, cow; GeneBank Y and W). (In parentheses are one-letter codes for amino NM 181036, horse; GeneBank NM 001081788, dog: Gen acids). Bank AB242566, wolf: GeneBank NM 001048031, It has been reported that a polypeptide having an amino chicken; GenBank NM 205522, and Zebrafish; GenBank acid sequence derived from an original amino acid sequence NM 198338. Thus, AE1 as described above can also be 60 by modification (Such as deletion, addition and/or substitu used. Other SLC4 anion exchangers can also be used since the tion of one or more amino acids) maintains the biological sequence information thereof has been registered in various activity of the original polypeptide (Mark, D. F. et al., Proc. databases. Natl. Acad. Sci. USA (1984) 81, 5662-5666; Zoller, M. J. & The polypeptide which has an amino acid sequence derived Smith, M. Nucleic Acids Research (1982) 10, 6487-6500; from the amino acid sequence as shown in SEQID NO: 2 by 65 Wang, A. et al., Science 224, 1431-1433; Dalbadie-McFar Substitution, deletion, addition and/or insertion of one or land, Get al., Proc. Natl. Acad. Sci. USA (1982) 79, 6409 more amino acid residues and yet has SLC4 anion exchanger 6413). US 9,068,212 B2 10 As one example of the polypeptide in which one or more W.J. and Lipman, D. J., Proc. Natl. Acad. Sci. USA (1983) amino acid residues are added to the SLC4 anion exchanger 80, 726-730 may be followed. derived from human or the like, a fusion polypeptide com The polypeptide may vary in amino acid sequence, prising the SLC4 anion exchanger derived from human or the molecular weight, isoelectric point, presence or absence of like may be given. Such a fusion polypeptide is composed of 5 Sugar chains, morphology, etc. depending on the cell or host the SLC4 anion exchanger derived from human or the like and that produce the polypeptide or the purification method that other polypeptide fused thereto. Such a fusion polypeptide will be described later. However, as long as the resultant may be prepared by linking a gene encoding the SLC4 anion polypeptide has functions equivalent to the functions of the exchanger derived from human or the like in frame with a SLC4 anion exchanger derived from human or the like, a gene encoding the other polypeptide, transferring the result 10 DNA encoding the polypeptide can be used in the present ant DNA into an expression vector and expressing the DNA in invention. For example, when the polypeptide of the present a host cell. Techniques known to those skilled in the art may invention is expressed in a prokaryote (e.g., Escherichia coli), be used. There is no limitation on the polypeptide to be fused a methionine reside is added to the N-terminus of the initial to the SLC4 anion exchanger derived from human or the like. 15 amino acid sequence of the polypeptide. When the polypep Examples of polypeptides to be fused to the SLC4 anion tide is expressed in a eukaryote (e.g., a mammalian cell), the exchanger derived from human or the like include, but are not N-terminal signal sequence is removed. These polypeptides limited to, FLAG (Hopp, T. P. et al., BioTechnology (1988) 6, can be used in the present invention. 1204-1210), 6xHis comprising six histidine (His) residues In the present invention, as a DNA encoding an SLC4 anion (SEQID NO:9), 10xHis (SEQID NO: 10), influenza hemag- 20 exchanger, a DNA having the nucleotide sequence as shown glutinin (HA), human c-myc fragment, VSV-GP fragment, in SEQID NO: 1 may be used. Alternatively, a DNA which p18HIV fragment, T7-tag HSV-tag, E-tag, SV40T antigen hybridizes to a DNA complementary to a DNA having the fragment, 1ck tag, C-tubulin fragment, B-tag, protein C frag nucleotide sequence as shown in SEQID NO: 1 under strin ment, glutathione-S-transferase (GST), influenza hemagglu gent conditions and yet encodes a polypeptide having SLC4 tinin (HA), immunoglobulin constant region, B-galactosidase 25 anion exchanger activity, may be used. SEQID NO. 1 shows and maltose-binding protein (MBP). the nucleotide sequence of human AE1. Aside from the A commercially available gene encoding Such polypeptide sequence information of human AE1, the counterpart infor is fused to the gene encoding the SLC4 anion exchanger mation about a mouse, a rat, a chimpanzee, a cow, a horse, a derived from human or the like. The fused gene thus prepared dog, a wolf, a chicken, a Zebrafish, and the like has been is expressed to prepare a fused polypeptide. 30 registered as mouse; GenBank NM 011403, rat; GeneBank An alternative method known to those skilled in the art for NM 012651, chimpanzee; GenBank XM 001151353, preparing polypeptides functionally equivalent to a specific cow; GeneBank NM 181036, horse; GeneBank polypeptide is a method using the hybridization technique NM 001081788, dog: GenBank AB242566, wolf: (Sambrook, J et al., Molecular Cloning 2nd ed., 9.47-9.58, GeneBank NM 001048031, chicken; GenBank Cold Spring Harbor Lab. Press, 1989). Those skilled in the art 35 NM 205522, and Zebrafish; GenBank NM 198338. Thus, could routinely isolate a DNA highly homologous to the DNA AE1 as described above can also be used. Other SLC4 anion sequence (e.g., SEQID NOS: 1) of the SLC4 anion exchanger exchangers can also be used since the sequence information derived from human or the like based on that DNA sequence thereof has been registered in various databases. or a part thereof, and isolate polypeptides functionally The DNA encoding an SLC4 anion exchanger can be used equivalent to the SLC4 anion exchanger derived from human 40 in the in vivo or invitro production of a desired polypeptide as or the like from that DNA. described above. Further, the DNA encoding an SLC4 anion Hybridization conditions for isolating a DNA encoding a exchanger may be used in the creation of a cell which strongly polypeptide functionally equivalent to the SLC4 anion expresses an SLC4 anion exchanger. The DNA encoding an exchanger derived from human or the like can be appropri SLC4 anion exchanger may take any form as long as it is ately selected by those skilled in the art. For example, low 45 capable of encoding an SLC4 anion exchanger. That is, the stringent hybridization conditions may be given. Low Strin DNA may be, for example, a cDNA synthesized from mRNA, gent hybridization conditions are, for example, 42° C. a genomic DNA or a chemically synthesized DNA. It should 2xSSC and 0.1% SDS, preferably 50° C., 2xSSC and 0.1% be noted that, as long as the DNA is capable of encoding an SDS. More preferably, high stringent conditions may be SLC4 anion exchanger, the DNA may have any nucleotide given. For example, high Stringent conditions are 65° C., 50 sequence based on the degeneracy of genetic codes. 2xSSC and 0.1% SDS. Under these conditions, as the hybrid The DNA encoding an SLC4 anion exchanger may be ization temperature is lowered, not only DNAs with high prepared by methods known to those skilled in the art. For homology but also DNAs with only low homology are example, the DNA may be prepared by preparing a cDNA obtained. Conversely, it is expected that only those DNAS library from a cell expressing an SLC4 anion exchanger and with high homology are obtained as the hybridization tem- 55 performing hybridization using a part of the DNA sequence perature is elevated. However, not only the temperature but ofan SLC4 anion exchanger (e.g., SEQID NO: 1) as a probe. also a plurality of factors (such as salt concentrations) affect The cDNA library may be prepared, for example, by the the stringency of hybridization. Those skilled in the art could method described in Sambrook, J. et al., Molecular Cloning, appropriately select these factors to realize similar stringency. Cold Spring Harbor Laboratory Press (1989). Alternatively, a The polypeptide encoded by a DNA isolated by these 60 commercial clNA library may be used. It is also possible to hybridization techniques may have 70% or more homology prepare the DNA encoding an SLC4 anion exchanger by and usually has high homology with the SLC4 anion preparing RNA from a cell expressing an SLC4 anion exchanger derived from human or the like in the amino acid exchanger, synthesizing oligo DNA molecules based on the sequence. The term “high homology” refers to usually 97% or DNA sequence of an SLC4 anion exchanger (e.g., SEQ ID more homology, preferably 98% or more homology, more 65 NO: 1), and performing PCR using the oligo DNA molecules preferably 99% or more homology. For determination of the as primers to thereby amplify a cDNA encoding an SLC4 homology of polypeptides, the algorithm described in Wilbur, anion exchanger. US 9,068,212 B2 11 12 Further, by determining the nucleotide sequence of the includes a DNA which encodes a polypeptide functionally resultant clNA, it is possible to determine the translation equivalent to the SLC4 anion exchanger derived from human region encoding an SLC4 anion exchanger and to obtain the or the like and has high identity with a DNA encoding the amino acid sequence of the SLC4 anion exchanger. Further, SLC4 anion exchanger derived from human or the like. The by Screening a genomic library using the resultant cDNA as a term “high identity” refers to usually 96% or more homology, probe, it is possible to isolate a genomic DNA. preferably 98% or more homology, more preferably 99% or Specifically, the following procedures may be used. First, more identity. The identity of nucleotide sequences may be mRNA is isolated from cells, tissues or the like expressing an determined by algorithm BLAST (Karlin and Altschul, Proc. SLC4 anion exchanger. For the isolation of mRNA, the total Natl. Acad. Sci. USA 90:5873-5877, 1993). Based on this RNA is prepared by known methods, for example, the guani 10 algorithm, programs such as BLASTN and BLASTX have dine ultracentrifugation method (Chirgwin, J. M. et al., Bio been developed (Altschul et al. J. Mol. Biol. 215:403-410, chemistry (1979) 18, 5294-5299), the AGPC method (Chom 1990). When nucleotide sequences are analyzed by BLASTN czynski, P. and Sacchi, N., Anal. Biochem. (1987) 162, 156 based on BLAST, parameters may be set as score=100 and 159) or the like, and then mRNA is purified from the total wordlength=12, for example. Specific procedures for these RNA using mRNA Purification Kit (Pharmacia), etc. Alter 15 analysis methods are known (.nlm.nih.gov.). natively, mRNA may be prepared directly using QuickPrep A bicarbonate transporter gene to be incorporated into a mRNA Purification Kit (Pharmacia). cell may be an SLC26 anion exchanger gene. Information of From the resultant mRNA, cDNA is synthesized using a a nucleotide sequence of an SLC26 anion exchanger gene and reverse transcriptase. Alternatively, cDNA may be synthe an amino acid encoded by the gene has been registered as sized using a kit such as AMV Reverse Transcriptase First GenBank AF331525 (human putative SLC26A9), GenBank Strand cDNA Synthesis Kit (SEIKAGAKU CORPORA NM 052934 (human SLC26A9 variant 1), GenBank TION). It is also possible to synthesize and amplify cloNA NM 134325 (human SLC26A9 variant 2), GenBank according to the 5'-RACE method (Frohman, M. A. et al., NM 1344.20(mouse SLC26A6), GenBank NM 177243 Proc. Natl. Acad. Sci. USA (1988) 85, 8998-9002; Belyay (mouse SLC26A9), GenBank AY24.0025 (Drosophila sky, A. et al., Nucleic Acids Res. (1989) 17, 2919-2932) using 25 Slc26d9702), GenBank AY240O23 (Drosophila 5'-Ampli FINDER RACE Kit (Clontech) and polymerase Slc26.d6928), GenBank AY240O22 (Drosophila chain reaction (PCR) with primers. Slc26.d6125), GenBank AY240O21 (Drosophila A DNA fragment of interest is prepared from the resultant Slc26.d5002), and GenBank AB084425 (eel Slc26A6). Thus, PCR product and ligated to a vector DNA to thereby prepare the SLC26 anion exchanger gene described as above can be a recombinant vector. The vector is introduced into a host 30 used. (e.g., E. coli), followed by selection of resultant colonies to The DNA encoding an SLC4 anion exchanger may be thereby obtain a desired recombinant vector. The nucleotide inserted into a vector. sequence of the DNA of interest may be confirmed by a When the host cell to be used is E. coli, it is preferable that known method such as the dideoxynucleotide chain termina the vector has a replication origin (“ori’) so that the vector is tion method. 35 largely amplified in E. coli (e.g., JM109, DH5O. HB101 and Further, a nucleotide sequence of higher expression effi XL1-Blue) and prepared in large quantity, and also genes for ciency can be designed for the DNA encoding an SLC4 anion selecting transformed E. coli (e.g., drug resistance genes that exchanger by considering the frequency of codon usage in the enable discrimination of transformant with some drugs such host to be used for expression (Grantham, R. et al., Nucleic as amplicillin, tetracycline, kanamycin or chloramphenicol). Acids Research (1981)9, p. 43-74). Further, the DNA encod 40 Examples of preferable vectors include, but are not limited to, ingan SLC4 anion exchanger can be modified using commer M13 vectors, puC vectors, pFBR322, pBluescript and pCR cially available kits or known methods. Examples of such Script. In addition to these vectors, pGEM-T, pIRECT, pT7. modifications include, but are not limited to, digestion with etc. may be enumerated when the vector is used for the pur restriction enzymes, insertion of synthetic oligonucleotides pose of subcloning a cDNA and cutting off the subcloned or appropriate DNA fragments, addition of linkers, and inser 45 cDNA. When the vector is used for the purpose of producing tion of an initiation codon (ATG) and/or a termination codon the polypeptide of the present invention, an expression vector (TAA, TGA or TAG). is especially useful. When expression in E. coli is intended, The DNA encoding an SLC4 anion exchanger also the expression vector preferably has the above-described fea includes a DNA which hybridizes to a DNA having the nucle tures so that the vector is amplified in E. coli, and it also otide sequence as shown in SEQ ID NO: 1 under stringent 50 preferably has a promoter which allows efficient expression conditions and encodes a polypeptide functionally equivalent in E. coli such as JM109, DH5C, HB101 or XL1-Blue, e.g., to an SLC4 anion exchanger. lacz promoter (Ward et al, Nature (1989) 341, 544-546: Stringent conditions can be appropriately selected by those FASEB.J. (1992) 6, 2422-2427), araB promoter (Better etal, skilled in the art, including, for example, low stringent con Science (1988) 240, 1041-1043) or T7 promoter. Specific ditions. Low Stringent conditions refer to, for example, 42 55 examples of Such vector include, in addition to those listed C., 2xSSC and 0.1% SDS, preferably 50° C., 2xSSC and above, pGEX-5x-1 (Pharmacia), QIAexpress system 0.1% SDS. More preferably, high stringent conditions may be (Qiagen), pFGFP, or pBT (for its host, T7 RNA polymerase selected. High stringent conditions refer to, for example, 65° expressing BL21 is preferred). C., 2xSSC and 0.1% SDS. Under these conditions, as the The vector may comprise signal sequences for polypeptide hybridization temperature is elevated, DNAs with a higher 60 secretion. When the polypeptide is to be produced in the homology can be obtained. The above-described DNA which periplasm of E. coli, pelB signal sequence (Lei, S. Pet al., J. hybridizes is preferably a DNA derived from nature, e.g., Bacteriol. (1987) 169, 4379) may be used as a signal sequence cDNA or chromosomal DNA. for polypeptide secretion. Introduction of the vector into a These DNAs isolated by hybridization techniques usually host cell may be performed, for example, by the calcium have a high nucleotide sequence identity with a DNA encod 65 chloride method or electroporation. ing the SLC4 anion exchanger derived from human or the In cases where a host cell other than E. coli is used, vectors like. The DNA encoding an SLC4 anion exchanger also useful for producing a desired polypeptide include, but are US 9,068,212 B2 13 14 not limited to, mammal-derived expression vectors e.g., polypeptide is not particularly limited. A gene encoding a pcDNA3 from Invitrogen; pEGF-BOS (Nucleic Acids. Res. desired polypeptide may be transferred after the transfer of a 1990, 18(17), p. 5322); pEF, pCDM8), insect cell-derived bicarbonate transporter gene. Alternatively, a bicarbonate expression vectors (e.g., Bac-to-BAC baculovairus expres transporter gene may be transferred after the transfer of a gene sion system from GIBCO BRL: pBacPAK8), plant-derived encoding a desired polypeptide. It is also possible to transfer expression vectors (e.g., pMH1, pMH2), animal virus-de a bicarbonate transporter gene and a gene encoding a desired rived expression vectors (e.g., pHSV, plMV, pAdexLcw), ret polypeptide simultaneously. rovirus-derived expression vectors (e.g., p7Ipneo), yeast-de A bicarbonate transporter gene and a gene encoding a rived expression vectors (e.g., Pichia Expression Kit from desired polypeptide may be transferred simultaneously in a Invitrogen; pNV 11; SP-Q01), and Bacillus subtilis-derived 10 single vector. Alternatively, they may be transferred sepa expression vectors (e.g., pPL608, pKTH5O). rately using a plurality of vectors. When expression of the polypeptide in animal cells (such Preferably, the cell which strongly expresses a bicarbonate as CHO cells, COS cells, NIH3T3 cells, etc.) is intended, the transporter further expresses cysteine Sulfinic acid decar vector preferably has a promoter necessary for expressing the boxylase (CSAD) or alanine aminotransferase (ALT) polypeptide in those cells. Examples of Such promoter 15 strongly in order to prepare a desired polypeptide. By trans include, but are not limited to, SV40 promoter (Mulligan etal, ferring a gene encoding the desired polypeptide into the cell Nature (1979) 277, 108), MMLV-LTR promoter, EF1C pro and culturing the resultant cell in a medium, the desired moter (Mizushima et al., Nucleic Acids Res. (1990) 18, polypeptide can be produced in a greater amount. 5322) and CMV promoter. More preferably, the vector also CSAD is originally known as an enzyme that converts has genes for selecting transformed cells (e.g., drug resistance alanine-3-sulfinic acid to hypotaurine. If cysteine Sulfinic genes that enable discrimination with drugs such as neomycin acid decarboxylase is strongly expressed in a CHO cell, the or G418). Examples of vectors having such properties cell Synthesizes an excess amount of B-alanine. include, but are not limited to, pMAM, p)R2, pFBK-RSV. A cell which strongly expresses CSAD is not particularly pBK-CMV, pCPRSV and pCP13. limited as long as the cell has an increased expression level of Further, when stable expression of a gene of interest and 25 CSAD compared to a corresponding natural cell. The natural intracellular amplification of the copy number of the gene are cell is not particularly limited. A cell which is used as a host indented, the following method may be used. Briefly, into in the production of a recombinant protein (e.g., CHO cells) CHO cells lacking a nucleic acid synthesis pathway, a vector may be used. having DHFR gene that complements the lack (e.g., pCHOI) As CSAD to be strongly expressed in a cell, CSAD derived is introduced, followed by amplification with methotrexate 30 from any organism may be used. Specifically, CSAD derived (MTX). On the other hand, when tentative expression of a from human, a rodent (such as mouse, rat orhamster), a puffer gene of interest is intended, a method may be used in which (such as Tiger puffer) or a sea squirt (such as Ciona intestina COS cells carrying a gene expressing SV40T antigen on the lis) may be used. Preferably, CSAD derived from human, a chromosome is transformed with a vector having the replica rodent or the same species as the host cell may be used. For tion origin of SV40 (e.g., pcD). As the replication origin, a 35 example, when the cell which is allowed to strongly express replication origin derived from polyomavirus, adenovirus or CSAD is Chinese hamsterovary cells (CHO cells), the CSAD bovine papillomavirus (BPV) may also be used. Further, the is preferably derived from human or hamster. expression vector may contain selectable markers for ampli As a cell which strongly expresses CSAD, a cell into which fying the copy number of the gene in a host cell system. a CSAD gene has been artificially transferred may be given. Examples of such selectable markers include, but are not 40 A cell into which a CSAD gene has been artificially trans limited to, aminoglycoside phosphotransferase (APH) gene, ferred can be prepared by methods known to those skilled in thymidine kinase (TK) gene, E. coli Xanthine-guanine phos the art. For example, such a cell may be prepared by incor phoribosyl transferase (Ecogpt) gene and dihydrofolate porating a CSAD gene into a vector and transforming the reductase (dhfr) gene. vector into a cell. Furthermore, the concept of “cells into The host cell into which the DNA encoding a bicarbonate 45 which a CSAD gene has been artificially transferred encom transporter (which may be incorporated in a vector) is trans passes herein cells in which an endogenous CSAD gene has ferred is not particularly limited. For example, E. coli or been activated by gene activation technology (see, for various animal cells may be used. If a DNA encoding a example, International Publication WO94/12650) so that desired polypeptide is transferred into a host cell into which a CSAD is strongly expressed. DNA encoding a bicarbonate transporter is transferred, this 50 As a CSAD gene to be transferred in a cell, any one of the host cell can express the bicarbonate transporter strongly, following DNAs (a1) to (e1) may be used. which leads to an increased production of the desired (a1) a DNA encoding a polypeptide having the amino acid polypeptide. Into the host cell into which a DNA encoding a sequence as shown in SEQ ID NO. 4 or the amino acid bicarbonate transporter is transferred, a DNA encoding sequence of UniProt Knowledgebase (Swiss-Prot and CSAD or ALT (which may be incorporated into a vector) may 55 TrEMBL) rat CSAD (Q64611), mouse CSAD (Q9 DBEO) or be further transferred. By transferring a DNA encoding a human CSAD (Q9Y600); desired polypeptide and a DNA encoding CSAD or ALT into (b1) a DNA encoding a polypeptide which has an amino acid a host cell into which a DNA encoding a bicarbonate trans sequence derived from the amino acid sequence as shown in porter is transferred, the yield of the desired polypeptide can SEQID NO. 4 or the amino acid sequence of UniProt Knowl be increased. For the production of the polypeptide, there are 60 edgebase (Swiss-Prot and TrEMBL) rat CSAD (Q64611), in vivo and in vitro production systems. Examples of in vitro mouse CSAD (Q9 DBEO) or human CSAD (Q9Y600) by production systems include systems using eukaryotes and Substitution, deletion, addition and/or insertion of one or systems using prokaryotes. more amino acid residues and yet has CSAD activity; When a desired polypeptide is produced using a cell into (c1) a DNA encoding a polypeptide having 70% or more which a bicarbonate transporter gene has been artificially 65 amino acid sequence homology with the amino acid sequence transferred, the order of the transfer of a bicarbonate trans as shown in SEQ ID NO. 4 or the amino acid sequence of porter gene and the transfer of a gene encoding a desired UniProt Knowledgebase (Swiss-Prot and TrEMBL) rat US 9,068,212 B2 15 16 CSAD (Q64611), mouse CSAD (Q9 DBE0) or human of one or more amino acids, preferably 1-30 amino acids, CSAD (Q9Y600) and yet having CSAD activity: more preferably 1-10 amino acids; a polypeptide having an (d1) a DNA having the nucleotide sequence as shown in SEQ amino acid sequence derived from the amino acid sequence of ID NO: 3 or the nucleotide sequence of GenBank rat CSAD CSAD derived from hamster or the like by addition of one or NM 021750, mouse CSAD NM 144942 or human CSAD more amino acids, preferably 1-30 amino acids, more prefer NM 015989; ably 1-10 amino acids; and a polypeptide having an amino (e1) a DNA which hybridizes to a DNA complementary to a acid sequence derived from the amino acid sequence of DNA having the nucleotide sequence as shown in SEQ ID CSAD derived from hamster or the like by substitution of one NO: 3 or the nucleotide sequence of GenBank rat CSAD or more amino acids, preferably 1-30 amino acids, more NM 021750, mouse CSAD NM 144942 or human CSAD 10 preferably 1-10 amino acids, with other amino acids. NM 015989 under stringent conditions and yet encodes a Amino acid residues to be mutated are not particularly polypeptide having CSAD activity. limited. Preferably, amino acid residues are mutated to other The concept of a CSAD activity encompasses an activity to amino acids in which the nature of the initial amino acid side catalyze 3-sulfino-L-alanine hypotaurine--CO for decar chain is conserved. Specific examples of the nature of amino boxylation. It is also an activity to decarboxylate L-cysteic 15 acid side chain include hydrophobic amino acids (A, I, L., M. acid. (EC-Number 4.1.1.29). F. P. W. Yand V), hydrophilic amino acids (R, D., N, C, E, Q, The CSAD activity can be measured as follows. C, H, K S and T), amino acids with an aliphatic side chain (G As taught by Davis K. et al., J Biomed Sci 2001: 8:359-364, A. V. L. I and P), amino acids with a hydroxyl group-contain '''CO produced from L-1-''Clcysteic acid by a decarboxy ing side chain (S. T and Y), amino acids with a Sulfur atom lase activity of CSAD is quantitated. containing side chain (C and M), amino acids with a carboxy In the present invention, a DNA encoding a polypeptide lic acid and amide-containing side chain (D, N., E and Q), having the amino acid sequence of SEQ ID NO. 4 or the amino acids with a base-containing side chain (R, K and H) amino acid sequence of UniProt Knowledgebase (Swiss-Prot and amino acids with an aromatic-containing side chain (H. F. and TrEMBL) rat CSAD (Q64611), mouse CSAD Y and W). (In parentheses are one-letter codes for amino (Q9DBE0) or human CSAD (Q9Y600) may be used as a 25 acids). DNA encoding CSAD. Besides that, a DNA encoding a It has been reported that a polypeptide having an amino polypeptide having the amino acid sequence of SEQID NO: acid sequence derived from an original amino acid sequence 4 or the amino acid sequence of UniProt Knowledgebase by modification (Such as deletion, addition and/or substitu (Swiss-Prot and TrEMBL) rat CSAD (Q64611), mouse tion of one or more amino acids) maintains the biological CSAD (Q9 DBEO) or human CSAD (Q9Y600) in which 30 activity of the original polypeptide (Mark, D. F. et al., Proc. one or a plurality of amino acid(s) is/are substituted, deleted, Natl. Acad. Sci. USA (1984) 81, 5662-5666; Zoller, M. J. & added, and/or inserted, and also having CSAD activity may Smith, M. Nucleic Acids Research (1982) 10, 6487-6500; be used. Wang, A. et al., Science 224, 1431-1433; Dalbadie-McFar The polypeptide having the amino acid sequence of SEQ land, Get al., Proc. Natl. Acad. Sci. USA (1982) 79, 6409 ID NO. 4 or the amino acid sequence of UniProt Knowledge 35 6413). base (Swiss-Prot and TrEMBL) rat CSAD (Q64611), mouse As one example of the polypeptide in which one or more CSAD (Q9 DBEO) or human CSAD (Q9Y600) in which amino acid residues are added to CSAD derived from hamster one or a plurality of amino acid(s) is/are substituted, deleted, or the like, a fusion polypeptide comprising CSAD derived added, and/or inserted, and also having CSAD activity is from hamster or the like may be given. Such a fusion polypep functionally equivalent to CSAD derived from hamster, rat, 40 tide is composed of CSAD derived from hamster or the like mouse or human (hereinafter sometimes referred to as and other polypeptide fused thereto. Such a fusion polypep “CSAD derived from hamster or the like). Such a polypeptide tide may be prepared by linking a gene encoding CSAD encompasses, for example, mutants of CSAD derived from derived from hamster or the like in frame with a gene encod hamster or the like. ing the other polypeptide, transferring the resultant DNA into As methods well-known to those skilled in the art for 45 an expression vector and expressing the DNA in a host cell. preparing polypeptides functionally equivalent to a specific Techniques known to those skilled in the art may be used. polypeptide, methods of introducing mutations into polypep There is no limitation on the polypeptide to be fused to CSAD tides may be given. For example, those skilled in the art could derived from hamster or the like. prepare polypeptides functionally equivalent to CSAD Examples of polypeptides to be fused to CSAD derived derived from hamster or the like by appropriately introducing 50 from hamster or the like include, but are not limited to, FLAG mutations into amino acids of CSAD derived from hamster or (Hopp, T. P. et al., BioTechnology (1988) 6, 1204-1210), the like by site-directed mutagenesis (Hashimoto-Gotoh, T. et 6xHis comprising six histidine (His) residues (SEQ ID NO: al. (1995) Gene 152, 271-275: Zoller, MJ, and Smith, M. 9), 10xHis (SEQID NO: 10, influenza hemagglutinin (HA), (1983) Methods Enzymol. 100, 468-500; Kramer, W. et al. human c-myc fragment, VSV-GP fragment, p.18HIV frag (1984) Nucleic Acids Res. 12,9441-9456; Kramer W, and 55 ment, T7-tag, HSV-tag, E-tag, SV40T antigen fragment, 1ck Fritz HJ (1987) Methods. Enzymol. 154,350-367; Kunkel, T tag, C-tubulin fragment, B-tag, protein C fragment, glu A (1985) Proc Natl Acad Sci USA. 82, 488-492: Kunkel tathione-S-transferase (GST), influenza hemagglutinin (HA), (1988) Methods Enzymol. 85, 2763-2766). Mutations in immunoglobulin constant region, B-galactosidase and mal amino acids may also occur in nature. tose-binding protein (MBP). Specific examples of polypeptides functionally equivalent 60 A commercially available gene encoding Such polypeptide to CSAD derived from hamster or the like include, but are not is fused to the gene encoding CSAD derived from hamster or limited to, a polypeptide having an amino acid sequence the like. The fused gene thus prepared is expressed to prepare derived from the amino acid sequence of CSAD derived from a fused polypeptide. hamster or the like (e.g., the amino acid sequence of SEQID An alternative method known to those skilled in the art for NO. 4 or the amino acid sequence of UniProt Knowledgebase 65 preparing polypeptides functionally equivalent to a specific (Swiss-Prot and TrEMBL) rat CSAD (Q64611), mouse polypeptide is a method using the hybridization technique CSAD (Q9 DBE0) or human CSAD (Q9Y600)) by deletion (Sambrook, J et al., Molecular Cloning 2nd ed., 9.47-9.58, US 9,068,212 B2 17 18 Cold Spring Harbor Lab. Press, 1989). Those skilled in the art as it is capable of encoding CSAD. That is, the DNA may be, could routinely isolate a DNA highly homologous to the DNA for example, a cDNA synthesized from mRNA, a genomic sequence of CSAD derived from hamster or the like (e.g., the DNA or a chemically synthesized DNA. It should be noted DNA sequence of SEQ ID NO: 3 or the DNA sequence of that, as long as the DNA is capable of encoding CSAD, the GenBank rat CSAD NM 021750, mouse CSAD 5 DNA may have any nucleotide sequence based on the degen NM 144942 or human CSAD NM 015989), based on that eracy of genetic codes. DNA sequence or a part thereof, and isolate polypeptides The DNA encoding CSAD may be prepared by methods functionally equivalent to CSAD derived from hamster or the known to those skilled in the art. For example, the DNA may like from that DNA. be prepared by preparing a cDNA library from a cell express Hybridization conditions for isolating a DNA encoding a 10 ing CSAD and performing hybridization using a part of the polypeptide functionally equivalent to CSAD derived from DNA sequence of CSAD (e.g., the nucleotide sequence of hamster or the like can be appropriately selected by those SEQ ID NO: 3 or the nucleotide sequence of GenBank rat skilled in the art. For example, low stringent hybridization CSAD NM 021750, mouse CSAD NM 144942 or human conditions may be given. Low Stringent hybridization condi CSAD NM 015989) as a probe. The cDNA library may be tions are, for example, 42°C., 2xSSC and 0.1% SDS, pref 15 prepared, for example, by the method described in Sambrook, erably 50° C., 2xSSC and 0.1% SDS. More preferably, high J. et al., Molecular Cloning, Cold Spring Harbor Laboratory stringent conditions may be given. For example, high Strin Press (1989). Alternatively, a commercial cDNA library may gent conditions are 65° C., 2xSSC and 0.1% SDS. Under be used. It is also possible to prepare the DNA encoding these conditions, as the hybridization temperature is lowered, CSAD by preparing RNA from a cell expressing CSAD, not only DNAs with high homology but also DNAs with only synthesizing oligo DNA molecules based on the DNA low homology are obtained. Conversely, it is expected that sequence of CSAD (e.g., the nucleotide sequence of SEQID only those DNAs with high homology are obtained as the NO: 3 or the nucleotide sequence of GenBank rat CSAD hybridization temperature is elevated. However, not only the NM 021750, mouse CSAD NM 144942 or human CSAD temperature but also a plurality of factors (such as salt con NM 015989), and performing PCR using the oligo DNA centrations) affect the stringency of hybridization. Those 25 molecules as primers to thereby amplify a cDNA encoding skilled in the art could appropriately select these factors to CSAD. realize similar stringency. Further, by determining the nucleotide sequence of the The polypeptide encoded by a DNA isolated by these resultant clNA, it is possible to determine the translation hybridization techniques may have 70% or more homology region encoding the polypeptide and to obtain the amino acid and usually has high homology with CSAD derived from 30 sequence of CSAD. Further, by Screening a genomic library hamster or the like in the amino acid sequence. The term using the resultant cDNA as a probe, it is possible to isolate a “high homology” refers to usually 97% or more homology, genomic DNA. preferably 98% or more homology, more preferably 99% or Specifically, the following procedures may be used. First, more homology. For determination of the homology of mRNA is isolated from cells, tissues or the like expressing polypeptides, the algorithm described in Wilbur, W. J. and 35 CSAD. For the isolation of mRNA, the total RNA is prepared Lipman, D.J., Proc. Natl. Acad. Sci. USA (1983) 80,726-730 by known methods, for example, the guanidine ultracentrifu may be followed. gation method (Chirgwin, J. M. et al., Biochemistry (1979) The polypeptide may vary in amino acid sequence, 18, 5294-5299), the AGPC method (Chomczynski, P. and molecular weight, isoelectric point, presence or absence of Sacchi, N., Anal. Biochem. (1987) 162, 156-159) or the like, Sugar chains, morphology, etc. depending on the cell or host 40 and then mRNA is purified from the total RNA using mRNA that produce the polypeptide or the purification method that Purification Kit (Pharmacia), etc. Alternatively, mRNA may will be described later. However, as long as the resultant be prepared directly using QuickPrep mRNA Purification Kit polypeptide has functions equivalent to the functions of (Pharmacia). CSAD derived from hamster or the like, a DNA encoding the From the resultant mRNA, cDNA is synthesized using a polypeptide can be used in the present invention. For 45 reverse transcriptase. Alternatively, cDNA may be synthe example, when the polypeptide is expressed in a prokaryote sized using a kit such as AMV Reverse Transcriptase First (e.g., Escherichia coli), a methionine reside is added to the Strand cDNA Synthesis Kit (SEIKAGAKU CORPORA N-terminus of the initial amino acid sequence of the polypep TION). It is also possible to synthesize and amplify cDNA tide. When the polypeptide is expressed in a eukaryote (e.g., according to the 5'-RACE method (Frohman, M. A. et al., a mammalian cell), the N-terminal signal sequence is 50 Proc. Natl. Acad. Sci. USA (1988)85,8998-9002; Belyavsky, removed. A DNA encoding Such a polypeptide can be used in A. et al., Nucleic Acids Res. (1989) 17, 2919-2932) using the present invention. 5'-Ampli FINDER RACE Kit (Clontech) and polymerase In the present invention, a DNA having the nucleotide chain reaction (PCR) with primers. sequence of SEQ ID NO: 3 or the nucleotide sequences of A DNA fragment of interest is prepared from the resultant GenBank rat CSAD NM 021750, mouse CSAD 55 PCR product and ligated to a vector DNA to thereby prepare NM 144942, or human CSADNM 015989 may be used as a recombinant vector. The vector is introduced into a host a DNA that encodes CSAD. Besides that, a DNA encoding a (e.g., E. coli), followed by selection of resultant colonies to polypeptide hybridizing with a DNA complementary to DNA thereby obtain a desired recombinant vector. The nucleotide having the nucleotide sequence of SEQ ID NO: 3 or the sequence of the DNA of interest may be confirmed by a nucleotide sequences of GenBank rat CSAD NM 021750, 60 known method Such as the dideoxynucleotide chain termina mouse CSAD NM 144942, or human CSAD NM 015989 tion method. under a stringent condition, and also having CSAD activity Further, a nucleotide sequence of a higher expression effi may be used. ciency can be designed for the DNA encoding CSAD by The DNA encoding CSAD is used to prepare a cell which considering the frequency of codon usage in the host to be strongly expresses CSAD and thereafter used in the in vivo or 65 used for expression (Grantham, R. et al., Nucleic Acids in vitro production of a desired polypeptide as described Research (1981) 9, p. 43-74). Further, the DNA encoding above. The DNA encoding CSAD may take any form as long CSAD can be modified using commercially available kits or US 9,068,212 B2 19 20 known methods. Examples of such modifications include, but As ALT to be strongly expressed in a cell. ALT derived are not limited to, digestion with restriction enzymes, inser from any organism may be used. Specifically, ALTs derived tion of synthetic oligonucleotides or appropriate DNA frag from human, mouse, rat, dog, African clawed frog, fruit fly, ments, addition of linkers, and insertion of an initiation codon nematode, Japanese rice, Cyanidioschyzon merolae, Saccha (ATG) and/or a termination codon (TAA, TGA or TAG). romyces cerevisiae, Ashbya gossypii, Candida albicans, The DNA encoding CSAD also includes a DNA which Schizosaccharomyces pombe, Aspergillus nidulans, hybridizes to a DNA complementary to a DNA having the Aspergillus fumigatus, Aspergillus Oryzae, Cryptococcus nucleotide sequence as shown in SEQID NO: 3 or the nucle neoformans, Dictyostelium discoideum, Trypanosoma bru otide sequence of GenBank rat CSAD NM 021750, mouse cei, Leishmania major; Entamoeba histolytica and Trypano CSAD NM 144942 or human CSAD NM O15989 under 10 soma Cruzi are known and can be used. Preferably, ALT stringent conditions and encodes a polypeptide functionally derived from human, a rodent or the same species as the host equivalent to CSAD. cell may be used. For example, when the cell which is allowed Stringent conditions can be appropriately selected by those to strongly express ALT is Chinese hamster ovary cells (CHO skilled in the art, including, for example, low stringent con cells), ALT is preferably derived from human or hamster. For ditions. Low Stringent conditions refer to, for example, 42 15 ALT in humans, mice, and yeast, variants (ALT1 and ALT2) C., 2xSSC and 0.1% SDS, preferably 50° C., 2xSSC and exist. ALT2 has 80% or greater homology to ALT1 at the 0.1% SDS. More preferably, high stringent conditions may be amino acid level. ALT1 was forcedly expressed in the selected. High stringent conditions refer to, for example, 65° Examples and Referential Examples described later. C., 2xSSC and 0.1% SDS. Under these conditions, as the As an ALT gene to be strongly expressed in a cell, any one hybridization temperature is elevated, DNAs with a higher of the following DNAs (a2) to (e2) encoding ALT may be homology can be obtained. The above-described DNA which used. hybridizes is preferably a DNA derived from nature, e.g., (a2) a DNA encoding a polypeptide having the amino acid cDNA or chromosomal DNA. sequence of KEGG/ENZYME: 2.6.1.2/Homo sapiens (hu These DNAs isolated by hybridization techniques usually man): 2875, KEGG/ENZYME: 2.6.1.2/Homo sapiens (hu have a high nucleotide sequence identity with a DNAencod 25 man): 84706, KEGG/ENZYME: 2.6.1.2/Mus musculus ing CSAD derived from hamster or the like. The DNA encod (mouse): 76282, KEGG/ENZYME: 2.6.1.2/Mus musculus ing CSAD also includes a DNA which encodes a polypeptide (mouse): 108682, KEGG/ENZYME: 2.6.1.2/Rattus norvegi functionally equivalent to CSAD derived from hamster or the cus (rat): 81670, KEGG/ENZYME: 2.6.1.2/Canis familiaris like and has high identity with a DNA encoding CSAD (dog): 609510, KEGG/ENZYME: 2.6.1.2/Xenopus laevis derived from hamster or the like. The term “high identity” 30 (African clawed frog): 444533, KEGG/ENZYME: 2.6.1.2/ refers to usually 96% or more homology, preferably 98% or Drosophila melanogaster (fruit fly): Dmel CG1640, KEGG/ more homology, more preferably 99% or more identity. The ENZYME: 2.6.1.2/Caenorhabditis elegans (nematode): identity of nucleotide sequences may be determined by algo C32F10.8, KEGG/ENZYME: 2.6.1.2/Oryza sativa japonica rithm BLAST (Karlin and Altschul, Proc. Natl. Acad. Sci. (Japanese rice): 4342210, KEGG/ENZYME: 2.6.1.2/Oryza USA 90:5873-5877, 1993). Based on this algorithm, pro 35 sativa japonica (Japanese rice): 4348524. KEGG/ENZYME: grams such as BLASTN and BLASTX have been developed 2.6.1.2/Cyanidioschyzon merolae: CMM066C, KEGG/EN (Altschul et al. J. Mol. Biol. 215:403-410, 1990). When ZYME: 2.6.1.2/Saccharomyces cerevisiae: YLR089c, nucleotide sequences are analyzed by BLASTN based on KEGG/ENZYME: 2.6.1.2/Saccharomyces cerevisiae: BLAST, parameters may be set as score=100 and YDR111c, KEGG/ENZYME: 2.6.1.2/Ashbya gossypii (Er wordlength=12, for example. Specific procedures for these 40 emothecium gossypii): AGOS AGR085W KEGG/EN analysis methods are known (ncbi.nlm.nih.gov.). ZYME: 2.6.1.2/Candida albicans: CaO19 346, KEGG/EN ALT is fundamentally known as an enzyme that produces ZYME: 2.6.1.2/Schizosaccharomyces pombe: SPBC582.08, glutamate by transferring an amino group from alanine to KEGG/ENZYME: 2.6.1.2/Aspergillus nidulans: AN1923.2, 2-oxoglutarate. If the reaction of biosynthesizing pyruvate KEGG/ENZYME: 2.6.1.2/Aspergillus fumigatus: AFUA and glutamate from alanine could be promoted by strongly 45 6G07770, KEGG/ENZYME: 2.6.1.2/Aspergillus oryzae: expressing ALT in host cells such as CHO cells, the products AO090003000164, KEGG/ENZYME: 2.6.1.2/Cryptococcus might be utilized in metabolism during a TCA cycle and neoformans JEC21: CNG01490, KEGG/ENZYME: 2.6.1.2/ glucose production by glycogenesis, and this might improve Dictyostelium discoideum: DDB 0232139, KEGG/EN cell culture behavior, leading to high-yield production of the ZYME: 2.6.1.2/Trypanosoma brucei: Tb927.1.3950, KEGG/ desired polypeptide. 50 ENZYME: 2.6.1.2/Leishmania major: LimjF12.0630, The strongly ALT expressing cells are not particularly lim KEGG/ENZYME: 2.6.1.2/Entamoeba histolytica. ited as long as they are capable of ALT expression at higher 233.t00009, KEGG/ENZYME: 2.6.1.2/Entamoeba his levels than natural cells. Natural cells include, but are not tolytica. 24.t00016, KEGG/ENZYME: 2.6.1.2/Trypano particularly limited to, cells that are used as hosts in the soma Cruzi: 506529.420, KEGG/ENZYME: 2.6.1.2/Trypa production of recombinant proteins and may be exemplified 55 nosoma cruzi: 506529.430, KEGG/ENZYME: 2.6.1.2/ by CHO cells. Trypanosoma cruzi: 510889.120 or KEGG/ENZYME: As a cell which strongly expresses ALT, a cell into which an 2.6.1.2/Trypanosoma cruzi: 510889.140; ALT gene has been artificially transferred may be given. A (b2) a DNA encoding a polypeptide which has an amino cell into which an ALT gene has been artificially transferred acid sequence derived from the amino acid sequence of can be prepared by methods known to those skilled in the art. 60 KEGG/ENZYME: 2.6.1.2/Homo sapiens (human): 2875, For example, such a cell may be prepared by incorporating an KEGG/ENZYME: 2.6.1.2/Homo sapiens (human): 84706, ALT gene into a vector and transforming the vector into a cell. KEGG/ENZYME: 2.6.1.2/Mus musculus (mouse): 76282, Furthermore, the concept of “cells into which an ALT gene KEGG/ENZYME: 2.6.1.2/Mus musculus (mouse): 108682, has been artificially transferred encompasses herein cells in KEGG/ENZYME: 2.6.1.2/Rattus norvegicus (rat): 81670, which an endogenous ALT gene has been activated by gene 65 KEGG/ENZYME: 2.6.1.2/Canis familiaris (dog): 6095.10, activation technology (see, for example, International Publi KEGG/ENZYME: 2.6.1.2/Xenopus laevis (African clawed cation WO94/12650) so that ALT is strongly expressed. frog): 444533, KEGG/ENZYME: 2.6.1.2/Drosophila mela US 9,068,212 B2 21 22 nogaster (fruit fly): Dmel CG1640, KEGG/ENZYME: (d2) a DNA having the nucleotide sequence of KEGG/ 2.6.1.2/Caenorhabditis elegans (nematode): C32F10.8, ENZYME: 2.6.1.2/Homo sapiens (human): 2875, KEGG/ KEGG/ENZYME: 2.6.1.2/Oryza sativa japonica (Japanese ENZYME: 2.6.1.2/Homo sapiens (human): 84706, KEGG/ rice): 4342210, KEGG/ENZYME: 2.6.1.2/Oryza sativa ENZYME: 2.6.1.2/Mus musculus (mouse): 76282, KEGG/ japonica (Japanese rice): 4348524, KEGG/ENZYME: ENZYME: 2.6.1.2/Mus musculus (mouse): 108682, KEGG/ 2.6.1.2/Cyanidioschyzon merolae: CMM066C, KEGG/EN ENZYME: 2.6.1.2/Rattus norvegicus (rat): 81670, KEGG/ ZYME: 2.6.1.2/Saccharomyces cerevisiae: YLR089c, ENZYME: 2.6.1.2/Canis familiaris (dog): 609510, KEGG/ KEGG/ENZYME: 2.6.1.2/Saccharomyces cerevisiae: ENZYME: 2.6.1.2/Xenopus laevis (African clawed frog): YDR111c, KEGG/ENZYME: 2.6.1.2/Ashbya gossypii (Er 444533, KEGG/ENZYME: 2.6.1.2/Drosophila melano emothecium gossypii): AGOS AGR085W, KEGG/EN 10 gaster (fruit fly): Dmel CG1640 KEGG/ENZYME: 2.6.1.2/ ZYME: 2.6.1.2/Candida albicans: CaO19 346, KEGG/EN Caenorhabditis elegans (nematode): C32F10.8, KEGG/EN ZYME: 2.6.1.2/Schizosaccharomyces pombe: SPBC582.08, ZYME: 2.6.1.2/Oryza sativa japonica (Japanese rice): KEGG/ENZYME: 2.6.1.2/Aspergillus nidulans: AN1923.2, 4342210, KEGG/ENZYME: 2.6.1.2/Oryza sativa japonica KEGG/ENZYME: 2.6.1.2/Aspergillus fumigatus: AFUA (Japanese rice): 4348524, KEGG/ENZYME: 2.6.1.2/Cyan 6G07770, KEGG/ENZYME: 2.6.1.2/Aspergillus oryzae: 15 idioschyzon merolae: CMM066C, KEGG/ENZYME: AO090003000164, KEGG/ENZYME: 2.6.1.2/Cryptococcus 2.6.1.2/Saccharomyces cerevisiae: YLR089c, KEGG/EN neoformans JEC21: CNG01490, KEGG/ENZYME: 2.6.1.2/ ZYME: 2.6.1.2/Saccharomyces cerevisiae: YDR111c, Dictyostelium discoideum: DDB 0232139, KEGG/EN KEGG/ENZYME: 2.6.1.2/Ashbya gossypii (Eremothecium ZYME: 2.6.1.2/Trypanosoma brucei: Tb927.1.3950, KEGG/ gossypii): AGOS AGR085W, KEGG/ENZYME: 2.6.1.2/ ENZYME: 2.6.1.2/Leishmania major: LimjF12.0630, Candida albicans: CaO19 346, KEGG/ENZYME: 2.6.1.2/ KEGG/ENZYME: 2.6.1.2/Entamoeba histolytica. Schizosaccharomyces pombe: SPBC582.08, KEGG/EN 233.t.00009, KEGG/ENZYME: 2.6.1.2/Entamoeba his ZYME: 2.6.1.2/Aspergillus nidulans: AN1923.2, KEGG/ tolytica: 24.t00016, KEGG/ENZYME: 2.6.1.2/Trypano ENZYME: 2.6.1.2/Aspergillus filmigatus: AFUA soma Cruzi: 506529.420, KEGG/ENZYME: 2.6.1.2/Trypa 6G07770, KEGG/ENZYME: 2.6.1.2/Aspergillus oryzae: nosoma Cruzi: 506529.430 KEGG/ENZYME: 2.6.1.2/ 25 AO090003000164, KEGG/ENZYME: 2.6.1.2/Cryptococcus Trypanosoma cruzi: 510889. 120 or KEGG/ENZYME: neoformans JEC21: CNG01490, KEGG/ENZYME: 2.6.1.2/ 2.6.1.2/Trypanosoma Cruzi: 510889.140 by substitution, Dictyostelium discoideum: DDB 0232139, KEGG/EN deletion, addition and/or insertion of one or more (e.g., sev ZYME: 2.6.1.2/Trypanosoma brucei: Tb927.1.3950, KEGG/ eral) amino acid residues and yet has ALT activity; ENZYME: 2.6.1.2/Leishmania major: LimjF12.0630, (c2) a DNA encoding a polypeptide having 70% or more 30 KEGG/ENZYME: 2.6.1.2/Entamoeba histolytica. amino acid sequence homology with the amino acid sequence 233.t00009, KEGG/ENZYME: 2.6.1.2/Entamoeba his of KEGG/ENZYME: 2.6.1.2/Homo sapiens (human): 2875, tolytica. 24t00016, KEGG/ENZYME: 2.6.1.2/Trypano KEGG/ENZYME: 2.6.1.2/Homo sapiens (human): 84706, soma Cruzi: 506529.420, KEGG/ENZYME: 2.6.1.2/Trypa KEGG/ENZYME: 2.6.1.2/Mus musculus (mouse): 76282, nosoma cruzi: 506529.430, KEGG/ENZYME: 2.6.1.2/ KEGG/ENZYME: 2.6.1.2/Mus musculus (mouse): 108682, 35 Trypanosoma cruzi: 510889.120 or KEGG/ENZYME: KEGG/ENZYME: 2.6.1.2/Rattus norvegicus (rat): 81670, 2.6.1.2/Trypanosoma cruzi: 510889.140; KEGG/ENZYME: 2.6.1.2/Canis familiaris (dog): 609510, (e2) a DNA which hybridizes to a DNA complementary to KEGG/ENZYME: 2.6.1.2/Xenopus laevis (African clawed a DNA having the nucleotide sequence of KEGG/ENZYME: frog): 444533, KEGG/ENZYME: 2.6.1.2/Drosophila mela 2.6.1.2/Homo sapiens (human): 2875, KEGG/ENZYME: nogaster (fruit fly): Dmel CG1640, KEGG/ENZYME: 40 2.6.1.2/Homo sapiens (human): 84706, KEGG/ENZYME: 2.6.1.2/Caenorhabditis elegans (nematode): C32F10.8, 2.6.1.2/Mus musculus (mouse): 76282, KEGG/ENZYME: KEGG/ENZYME: 2.6.1.2/Oryza sativa japonica (Japanese 2.6.1.2/Mus musculus (mouse): 108682, KEGG/ENZYME: rice): 4342210, KEGG/ENZYME: 2.6.1.2/Oryza sativa 2.6.1.2/Rattus norvegicus (rat): 81670, KEGG/ENZYME: japonica (Japanese rice): 4348524, KEGG/ENZYME: 2.6.1.2/Canis familiaris (dog): 609510, KEGG/ENZYME: 2.6.1.2/Cyanidioschyzon merolae: CMM066C, KEGG/EN 45 2.6.1.2/Xenopus laevis (African clawed frog): 444533, ZYME: 2.6.1.2/Saccharomyces cerevisiae: YLR089c, KEGG/ENZYME: 2.6.1.2/Drosophila melanogaster (fruit KEGG/ENZYME: 2.6.1.2/Saccharomyces cerevisiae: fly): Dmel CG1640, KEGG/ENZYME: 2.6.1.2/Caenorhab YDR111c, KEGG/ENZYME: 2.6.1.2/Ashbya gossypii (Er ditis elegans (nematode): C32F10.8, KEGG/ENZYME: emothecium gossypii): AGOS AGR085W, KEGG/EN 2.6.1.2/Oryza sativa japonica (Japanese rice): 4342210, ZYME: 2.6.1.2/Candida albicans: CaO19 346, KEGG/EN 50 KEGG/ENZYME: 2.6.1.2/Oryza sativa japonica (Japanese ZYME: 2.6.1.2/Schizosaccharomyces pombe: SPBC582.08, rice): 4348524, KEGG/ENZYME: 2.6.1.2/Cyanidioschyzon KEGG/ENZYME: 2.6.1.2/Aspergillus nidulans: AN1923.2, merolae: CMMO66C, KEGG/ENZYME: 2.6.1.2/Saccharo KEGG/ENZYME: 2.6.1.2/Aspergillus fumigatus: AFUA myces cerevisiae:YLR089c, KEGG/ENZYME: 2.6.1.2/Sac 6G07770, KEGG/ENZYME: 2.6.1.2/Aspergillus oryzae: charomyces cerevisiae: YDR111c, KEGG/ENZYME: AO090003000164, KEGG/ENZYME: 2.6.1.2/Cryptococcus 55 2.6.1.2/Ashbya gossypii (Eremothecium gossypii): neoformans JEC21: CNG01490, KEGG/ENZYME: 2.6.1.2/ AGOS AGR085W, KEGG/ENZYME: 2.6.1.2/Candida Dictyostelium discoideum: DDB 0232139, KEGG/EN albicans: CaO19 346, KEGG/ENZYME: 2.6.1.2/ ZYME: 2.6.1.2/Trypanosoma brucei: Tb927.1.3950, KEGG/ Schizosaccharomyces pombe: SPBC582.08, KEGG/EN ENZYME: 2.6.1.2/Leishmania major: LimjF12.0630, ZYME: 2.6.1.2/Aspergillus nidulans: AN1923.2, KEGG/ KEGG/ENZYME: 2.6.1.2/Entamoeba histolytica. 60 ENZYME: 2.6.1.2/Aspergillus filmigatus: AFUA 233.t.00009, KEGG/ENZYME: 2.6.1.2/Entamoeba his 6G07770, KEGG/ENZYME: 2.6.1.2/Aspergillus oryzae: tolytica: 24.t00016, KEGG/ENZYME: 2.6.1.2/Trypano AO090003000164, KEGG/ENZYME: 2.6.1.2/Cryptococcus soma Cruzi: 506529.420, KEGG/ENZYME: 2.6.1.2/Trypa neoformans JEC21: CNG01490, KEGG/ENZYME: 2.6.1.2/ nosoma cruzi: 506529.430, KEGG/ENZYME: 2.6.1.2/ Dictyostelium discoideum: DDB 0232139, KEGG/EN Trypanosoma cruzi: 510889. 120 or KEGG/ENZYME: 65 ZYME: 2.6.1.2/Trypanosoma brucei: Tb927.1.3950, KEGG/ 2.6.1.2/Trypanosoma Cruzi: 510889.140 and yet having ALT ENZYME: 2.6.1.2/Leishmania major: LimjF12.0630, activity; KEGG/ENZYME: 2.6.1.2/Entamoeba histolytica: US 9,068,212 B2 23 24 233.t.00009, KEGG/ENZYME: 2.6.1.2/Entamoeba his Xenopus laevis (African clawed frog): 444533, KEGG/ tolytica: 24.t00016, KEGG/ENZYME: 2.6.1.2/Trypano ENZYME: 2.6.1.2/Drosophila melanogaster (fruit fly): soma Cruzi: 506529.420, KEGG/ENZYME: 2.6.1.2/Trypa Dmel CG1640, KEGG/ENZYME: 2.6.1.2/Caenorhabditis nosoma cruzi: 506529.430, KEGG/ENZYME: 2.6.1.2/ elegans (nematode): C32F10.8, KEGG/ENZYME: 2.6.1.2/ Trypanosoma cruzi: 510889. 120 or KEGG/ENZYME: 5 Oryza sativa japonica (Japanese rice): 4342210, KEGG/EN 2.6.1.2/Trypanosoma Cruzi: 510889.140 under stringent con ZYME: 2.6.1.2/Oryza sativa japonica (Japanese rice): ditions and yet encodes a polypeptide having ALT activity. 4348524, KEGG/ENZYME: 2.6.1.2/Cyanidioschyzon mero The concept of an ALT activity encompasses an enzyme lae: CMM066C, KEGG/ENZYME: 2.6.1.2/Saccharomyces activity to catalyze transfer of an amino group between an cerevisiae: YLR089c, KEGG/ENZYME: 2.6.1.2/Saccharo amino acid and an O-keto acid. 10 myces cerevisiae:YDR111c, KEGG/ENZYME: 2.6.1.2/Ash The ALT activity can be measured as follows. bya gossypii (Eremothecium gossypii): AGOS AGR085W. An ALT activity level is determined by a reagent for auto KEGG/ENZYME: 2.6.1.2/Candida albicans: CaO19 346, mated analyzer for measuring alanine aminotransferase KEGG/ENZYME: 2.6.1.2/Schizosaccharomyces pombe: (Runpia liquid S-ALT, approval number SPBC582.08, KEGG/ENZYME: 2.6.1.2/Aspergillus nidu 20900AMZ005.97000) and the method taught by Rajamohan 15 lans: AN1923.2, KEGG/ENZYME: 2.6.1.2/Aspergillus F. et. al., Protein Expression and Purification (2006) 48. fumigatus: AFUA 6G07770, KEGG/ENZYME: 2.6.1.2/As 81-89. pergillus oryzae: AO090003000164, KEGG/ENZYME: In the present invention, as a gene encoding ALT, a DNA 2.6.1.2/Cryptococcus neoformans JEC21: CNGO1490, encoding a polypeptide having the amino acid sequence of KEGG/ENZYME: 2.6.1.2/Dictyostelium discoideum: KEGG/ENZYME: 2.6.1.2/Homo sapiens (human): 2875, 20 DDB 0232139, KEGG/ENZYME: 2.6.1.2/Trypanosoma KEGG/ENZYME: 2.6.1.2/Homo sapiens (human): 84706, brucei. Th927.1.3950, KEGG/ENZYME: 2.6.1.2/Leishma KEGG/ENZYME: 2.6.1.2/Mus musculus (mouse): 76282, nia major: LimjF12.0630, KEGG/ENZYME: 2.6.1.2/Entam KEGG/ENZYME: 2.6.1.2/Mus musculus (mouse): 108682, oeba histolytica. 233.t00009, KEGG/ENZYME: 2.6.1.2/En KEGG/ENZYME: 2.6.1.2/Rattus norvegicus (rat): 81670, tamoeba histolytica. 24.t00016, KEGG/ENZYME: 2.6.1.2/ KEGG/ENZYME: 2.6.1.2/Canis familiaris (dog): 609510, 25 Trypanosoma cruzi: 506529.420, KEGG/ENZYME: 2.6.1.2/ KEGG/ENZYME: 2.6.1.2/Xenopus laevis (African clawed Trypanosoma cruzi: 506529.430, KEGG/ENZYME: 2.6.1.2/ frog): 444533, KEGG/ENZYME: 2.6.1.2/Drosophila mela Trypanosoma cruzi: 510889.120 or KEGG/ENZYME: nogaster (fruit fly): Dmel CG1640, KEGG/ENZYME: 2.6.1.2/Trypanosoma Cruzi: 510889.140 by substitution, 2.6.1.2/Caenorhabditis elegans (nematode): C32F10.8, deletion, addition and/or insertion of one or more amino acid KEGG/ENZYME: 2.6.1.2/Oryza sativa japonica (Japanese 30 residues and yet has ALTactivity is functionally equivalent to rice): 4342210, KEGG/ENZYME: 2.6.1.2/Oryza sativa ALT derived from human, mouse, rat, dog, African clawed japonica (Japanese rice): 4348524, KEGG/ENZYME: frog, fruit fly, nematode, Japanese rice, Cyanidioschyzon 2.6.1.2/Cyanidioschyzon merolae: CMM066C, KEGG/EN merolae, Saccharomyces cerevisiae, Ashbya gossypii, Can ZYME: 2.6.1.2/Saccharomyces cerevisiae: YLR089c, dida albicans, Schizosaccharomyces pombe, Aspergillus KEGG/ENZYME: 2.6.1.2/Saccharomyces cerevisiae: 35 nidulans, Aspergillus filmigatus, Aspergillus Oryzae, Crypto YDR111c, KEGG/ENZYME: 2.6.1.2/Ashbya gossypii (Er coccus neoformans, Dictyostelium discoideum, Trypano emothecium gossypii): AGOS AGR085W, KEGG/EN Soma brucei, Leishmania major; Entamoeba histolytica or ZYME: 2.6.1.2/Candida albicans: CaO19 346, KEGG/EN Trypanosoma Cruzi (hereinafter sometimes referred to as ZYME: 2.6.1.2/Schizosaccharomyces pombe: SPBC582.08, *ALT derived from human or the like'). Such a polypeptide KEGG/ENZYME: 2.6.1.2/Aspergillus nidulans: AN1923.2, 40 encompasses, for example, mutants of ALT derived from KEGG/ENZYME: 2.6.1.2/Aspergillus fumigatus: AFUA human or the like. In Example and Referential Examples 6G07770, KEGG/ENZYME: 2.6.1.2/Aspergillus oryzae: described below, a mutant in which four out of 496 amino AO090003000164, KEGG/ENZYME: 2.6.1.2/Cryptococcus acids were replaced (R53S, Q72R, F286S and M332K) was neoformans JEC21: CNG01490, KEGG/ENZYME: 2.6.1.2/ used. Dictyostelium discoideum: DDB 0232139, KEGG/EN 45 As methods well-known to those skilled in the art for ZYME: 2.6.1.2/Trypanosoma brucei: Tb927.1.3950, KEGG/ preparing polypeptides functionally equivalent to a specific ENZYME: 2.6.1.2/Leishmania major: LimjF12.0630, polypeptide, methods of introducing mutations into polypep KEGG/ENZYME: 2.6.1.2/Entamoeba histolytica. tides may be given. For example, those skilled in the art could 233.t.00009, KEGG/ENZYME: 2.6.1.2/Entamoeba his prepare polypeptides functionally equivalent to ALT derived tolytica: 24.t00016, KEGG/ENZYME: 2.6.1.2/Trypano 50 from human or the like by appropriately introducing muta soma Cruzi: 506529.420, KEGG/ENZYME: 2.6.1.2/Trypa tions into amino acids of ALT derived from human or the like nosoma cruzi: 506529.430, KEGG/ENZYME: 2.6.1.2/ by site-directed mutagenesis (Hashimoto-Gotoh, T. et al. Trypanosoma cruzi: 510889. 120 or KEGG/ENZYME: (1995) Gene 152,271-275; Zoller, MJ, and Smith, M. (1983) 2.6.1.2/Trypanosoma Cruzi: 510889.140 may be used. Alter Methods Enzymol. 100, 468-500; Kramer, Wet al. (1984) natively, a DNA encoding a polypeptide which has an amino 55 Nucleic Acids Res. 12,9441-9456; Kramer W, and Fritz H J acid sequence derived from the amino acid sequence (1987) Methods. Enzymol. 154,350-367; Kunkel, TA (1985) described above by substitution, deletion, addition and/or Proc Natl AcadSci USA. 82,488-492: Kunkel (1988) Meth insertion of one or more amino acid residues and yet has ALT ods Enzymol. 85,2763-2766). Mutations in amino acids may activity may be used. also occur in nature. The polypeptide which has an amino acid sequence derived 60 Specific examples of polypeptides functionally equivalent from the amino acid sequence of KEGG/ENZYME: 2.6.1.2/ to the ALT derived from human or the like include, but are not Homo sapiens (human): 2875, KEGG/ENZYME: 2.6.1.2/ limited to, a polypeptide having an amino acid sequence Homo sapiens (human): 84706, KEGG/ENZYME: 2.6.1.2/ derived from the amino acid sequence (e.g., the amino acid Mus musculus (mouse): 76282, KEGG/ENZYME: 2.6.1.2/ sequence of KEGG/ENZYME: 2.6.1.2/Homo sapiens (hu Mus musculus (mouse): 108682, KEGG/ENZYME: 2.6.1.2/ 65 man): 2875, KEGG/ENZYME: 2.6.1.2/Homo sapiens (hu Rattus norvegicus (rat): 81670, KEGG/ENZYME: 2.6.1.2/ man): 84706, KEGG/ENZYME: 2.6.1.2/Mus musculus Canis familiaris (dog): 6095.10, KEGG/ENZYME: 2.6.1.2/ (mouse): 76282, KEGG/ENZYME: 2.6.1.2/Mus musculus US 9,068,212 B2 25 26 (mouse): 108682, KEGG/ENZYME: 2.6.1.2/Rattus norvegi As one example of the polypeptide in which one or more cus (rat): 81670, KEGG/ENZYME: 2.6.1.2/Canis familiaris amino acid residues are added to the ALT derived from human (dog): 609510, KEGG/ENZYME: 2.6.1.2/Xenopus laevis or the like, a fusion polypeptide comprising the ALT derived (African clawed frog): 444533, KEGG/ENZYME: 2.6.1.2/ from human or the like may be given. Such a fusion polypep Drosophila melanogaster (fruit fly): Dmel CG1640 KEGG/ tide is composed of the ALT derived from human or the like ENZYME: 2.6.1.2/Caenorhabditis elegans (nematode): and other polypeptide fused thereto. Such a fusion polypep C32F10.8, KEGG/ENZYME: 2.6.1.2/Oryza sativa japonica tide may be prepared by linking a gene encoding the ALT (Japanese rice): 4342210, KEGG/ENZYME: 2.6.1.2/Oryza derived from human or the like inframe with a gene encoding sativa japonica (Japanese rice): 4348524. KEGG/ENZYME: the other polypeptide, transferring the resultant DNA into an 2.6.1.2/Cyanidioschyzon merolae: CMM066C, KEGG/EN 10 expression vector and expressing the DNA in a host cell. ZYME: 2.6.1.2/Saccharomyces cerevisiae: YLR089c, Techniques known to those skilled in the art may be used. KEGG/ENZYME: 2.6.1.2/Saccharomyces cerevisiae: There is no limitation on the polypeptide to be fused to the YDR111c, KEGG/ENZYME: 2.6.1.2/Ashbya gossypii (Er ALT derived from human or the like. emothecium gossypii): AGOS AGR085W, KEGG/EN Examples of polypeptides to be fused to the ALT derived ZYME: 2.6.1.2/Candida albicans: CaO19 346, KEGG/EN 15 from human or the like include, but are not limited to, FLAG ZYME: 2.6.1.2/Schizosaccharomyces pombe: SPBC582.08, (Hopp, T. P. et al., BioTechnology (1988) 6, 1204-1210), KEGG/ENZYME: 2.6.1.2/Aspergillus nidulans: AN1923.2, 6xHis comprising six histidine (His) residues (SEQ ID NO: KEGG/ENZYME: 2.6.1.2/Aspergillus fumigatus: AFUA 9), 10xHis (SEQID NO: 10), influenza hemagglutinin (HA), 6G07770, KEGG/ENZYME: 2.6.1.2/Aspergillus oryzae: human c-myc fragment, VSV-GP fragment, p.18HIV frag AO090003000164, KEGG/ENZYME: 2.6.1.2/Cryptococcus ment, T7-tag, HSV-tag, E-tag, SV40T antigen fragment, 1ck neoformans JEC21: CNG01490, KEGG/ENZYME: 2.6.1.2/ tag, C-tubulin fragment, B-tag, protein C fragment, glu Dictyostelium discoideum: DDB 0232139, KEGG/EN tathione-S-transferase (GST), influenza hemagglutinin (HA), ZYME: 2.6.1.2/Trypanosoma brucei: Tb927.1.3950, KEGG/ immunoglobulin constant region, B-galactosidase and mal ENZYME: 2.6.1.2/Leishmania major: LimjF12.0630, tose-binding protein (MBP). KEGG/ENZYME: 2.6.1.2/Entamoeba histolytica. 25 A commercially available gene encoding Such polypeptide 233.t.00009, KEGG/ENZYME: 2.6.1.2/Entamoeba his is fused to the gene encoding the ALT derived from human or tolytica: 24.t00016, KEGG/ENZYME: 2.6.1.2/Trypano the like. The fused gene thus prepared is expressed to prepare soma Cruzi: 506529.420, KEGG/ENZYME: 2.6.1.2/Trypa a fused polypeptide. nosoma cruzi: 506529.430, KEGG/ENZYME: 2.6.1.2/ An alternative method known to those skilled in the art for Trypanosoma cruzi: 510889. 120 or KEGG/ENZYME: 30 preparing polypeptides functionally equivalent to a specific 2.6.1.2/Trypanosoma cruzi: 510889.140) of the ALT derived polypeptide is a method using the hybridization technique from human or the like by deletion of one or more amino (Sambrook, J et al., Molecular Cloning 2nd ed., 9.47-9.58, acids, preferably 1-30 amino acids, more preferably 1-10 Cold Spring Harbor Lab. Press, 1989). Those skilled in the art amino acids; a polypeptide having an amino acid sequence could routinely isolate a DNA highly homologous to the DNA derived from the amino acid sequence of the ALT derived 35 sequence (e.g., the DNA sequence of KEGG/ENZYME: from human or the like by addition of one or more amino 2.6.1.2/Homo sapiens (human): 2875, KEGG/ENZYME: acids, preferably 1-30 amino acids, more preferably 1-10 2.6.1.2/Homo sapiens (human): 84706, KEGG/ENZYME: amino acids; and a polypeptide having an amino acid 2.6.1.2/Mus musculus (mouse): 76282, KEGG/ENZYME: sequence derived from the amino acid sequence of the ALT 2.6.1.2/Mus musculus (mouse): 108682, KEGG/ENZYME: derived from human or the like by substitution of one or more 40 2.6.1.2/Rattus norvegicus (rat): 81670, KEGG/ENZYME: amino acids, preferably 1-30 amino acids, more preferably 2.6.1.2/Canis familiaris (dog): 609510, KEGG/ENZYME: 1-10 amino acids, with other amino acids. 2.6.1.2/Xenopus laevis (African clawed frog): 444533, Amino acid residues to be mutated are not particularly KEGG/ENZYME: 2.6.1.2/Drosophila melanogaster (fruit limited. Preferably, amino acid residues are mutated to other fly): Dmel CG1640, KEGG/ENZYME: 2.6.1.2/Caenorhab amino acids in which the nature of the initial amino acid side 45 ditis elegans (nematode): C32F10.8, KEGG/ENZYME: chain is conserved. Specific examples of the nature of amino 2.6.1.2/Oryza sativa japonica (Japanese rice): 4342210, acid side chain include hydrophobic amino acids (A, I, L., M. KEGG/ENZYME: 2.6.1.2/Oryza sativa japonica (Japanese F. P. W. Yand V), hydrophilic amino acids (R, D., N, C, E, Q, rice): 4348524, KEGG/ENZYME: 2.6.1.2/Cyanidioschyzon H. K. Sand T), amino acids with an aliphatic side chain (G. A. merolae: CMMO66C, KEGG/ENZYME: 2.6.1.2/Saccharo V. L. I and P), amino acids with a hydroxyl group-containing 50 myces cerevisiae:YLR089c, KEGG/ENZYME: 2.6.1.2/Sac side chain (S. T and Y), amino acids with a sulfur atom charomyces cerevisiae: YDR111c, KEGG/ENZYME: containing side chain (Cand M), amino acids with a carboxy 2.6.1.2/Ashbya gossypii (Eremothecium gossypii): lic acid and amide-containing side chain (D, N., E and Q), AGOS AGR085W, KEGG/ENZYME: 2.6.1.2/Candida amino acids with a base-containing side chain (R, K and H) albicans: CaO19 346, KEGG/ENZYME: 2.6.1.2/ and amino acids with an aromatic-containing side chain (H. F. 55 Schizosaccharomyces pombe: SPBC582.08, KEGG/EN Y and W). (In parentheses are one-letter codes for amino ZYME: 2.6.1.2/Aspergillus nidulans: AN1923.2, KEGG/ acids). ENZYME: 2.6.1.2/Aspergillus filmigatus: AFUA It has been reported that a polypeptide having an amino 6G07770, KEGG/ENZYME: 2.6.1.2/Aspergillus oryzae: acid sequence derived from an original amino acid sequence AO090003000164, KEGG/ENZYME: 2.6.1.2/Cryptococcus by modification (Such as deletion, addition and/or substitu 60 neoformans JEC21: CNG01490, KEGG/ENZYME: 2.6.1.2/ tion of one or more amino acids) maintains the biological Dictyostelium discoideum: DDB 0232139, KEGG/EN activity of the original polypeptide (Mark, D. F. et al., Proc. ZYME: 2.6.1.2/Trypanosoma brucei: Tb927.1.3950, KEGG/ Natl. Acad. Sci. USA (1984) 81, 5662-5666; Zoller, M. J. & ENZYME: 2.6.1.2/Leishmania major: LimjF12.0630, Smith, M. Nucleic Acids Research (1982) 10, 6487-6500; KEGG/ENZYME: 2.6.1.2/Entamoeba histolytica. Wang, A. et al., Science 224, 1431-1433; Dalbadie-McFar 65 233.t00009, KEGG/ENZYME: 2.6.1.2/Entamoeba his land, Get al., Proc. Natl. Acad. Sci. USA (1982) 79, 6409 tolytica. 24.t00016, KEGG/ENZYME: 2.6.1.2/Trypano 6413). soma Cruzi: 506529.420, KEGG/ENZYME: 2.6.1.2/Trypa US 9,068,212 B2 27 28 nosoma cruzi: 506529.430, KEGG/ENZYME: 2.6.1.2/ 2.6.1.2/Ashbya gossypii (Eremothecium gossypii): Trypanosoma cruzi: 510889. 120 or KEGG/ENZYME: AGOS AGR085W, KEGG/ENZYME: 2.6.1.2/Candida 2.6.1.2/Trypanosoma cruzi: 510889.140) of the ALT derived albicans: CaO19 346, KEGG/ENZYME: 2.6.1.2/ from human or the like based on that DNA sequence or a part Schizosaccharomyces pombe: SPBC582.08, KEGG/EN thereof, and isolate polypeptides functionally equivalent to ZYME: 2.6.1.2/Aspergillus nidulans: AN1923.2, KEGG/ the ALT derived from human or the like from that DNA. ENZYME: 2.6.1.2/Aspergillus filmigatus: AFUA Hybridization conditions for isolating a DNA encoding a 6G07770, KEGG/ENZYME: 2.6.1.2/Aspergillus oryzae: polypeptide functionally equivalent to the ALT derived from AO090003000164, KEGG/ENZYME: 2.6.1.2/Cryptococcus human or the like can be appropriately selected by those neoformans JEC21: CNG01490, KEGG/ENZYME: 2.6.1.2/ skilled in the art. For example, low stringent hybridization 10 Dictyostelium discoideum: DDB 0232139, KEGG/EN conditions may be given. Low Stringent hybridization condi ZYME: 2.6.1.2/Trypanosoma brucei: Tb927.1.3950, KEGG/ tions are, for example, 42°C., 2xSSC and 0.1% SDS, pref ENZYME: 2.6.1.2/Leishmania major: LimjF12.0630, erably 50° C., 2xSSC and 0.1% SDS. More preferably, high KEGG/ENZYME: 2.6.1.2/Entamoeba histolytica. stringent conditions may be given. For example, high Strin 233.t00009, KEGG/ENZYME: 2.6.1.2/Entamoeba his gent conditions are 65° C., 2xSSC and 0.1% SDS. Under 15 tolytica. 24.t00016, KEGG/ENZYME: 2.6.1.2/Trypano these conditions, as the hybridization temperature is lowered, soma Cruzi: 506529.420, KEGG/ENZYME: 2.6.1.2/Trypa not only DNAs with high homology but also DNAs with only nosoma cruzi: 506529.430, KEGG/ENZYME: 2.6.1.2/ low homology are obtained. Conversely, it is expected that Trypanosoma cruzi: 510889.120 or KEGG/ENZYME: only those DNAs with high homology are obtained as the 2.6.1.2/Trypanosoma Cruzi: 510889.140 may be used. Alter hybridization temperature is elevated. However, not only the natively, a DNA which hybridizes to a DNA complementary temperature but also a plurality of factors (such as salt con to a DNA having the nucleotide sequence described above centrations) affect the stringency of hybridization. Those under Stringent conditions and yet encodes a polypeptide skilled in the art could appropriately select these factors to having ALT activity, may be used. realize similar stringency. The DNA encoding ALT can be used in the in vivo or in The polypeptide encoded by a DNA isolated by these 25 vitro production of a desired polypeptide as described above. hybridization techniques may have 70% or more homology Further, the DNA encoding ALT may be used in the creation and usually has high homology with the ALT derived from of a cell which strongly expresses ALT. The DNA encoding human or the like in the amino acid sequence. The term “high ALT may take any form as long as it is capable of encoding homology” refers to usually 97% or more homology, prefer ALT. That is, the DNA may be, for example, a cDNA synthe ably 98% or more homology, more preferably 99% or more 30 sized from mRNA, a genomic DNA or a chemically synthe homology. For determination of the homology of polypep sized DNA. It should be noted that, as long as the DNA is tides, the algorithm described in Wilbur, W.J. and Lipman, D. capable of encoding ALT, the DNA may have any nucleotide J., Proc. Natl. Acad. Sci. USA (1983) 80, 726-730 may be sequence based on the degeneracy of genetic codes. followed. The DNA encoding ALT may be prepared by methods The polypeptide may vary in amino acid sequence, 35 known to those skilled in the art. For example, the DNA may molecular weight, isoelectric point, presence or absence of be prepared by preparing a cDNA library from a cell express Sugar chains, morphology, etc. depending on the cell or host ing ALT and performing hybridization using a part of the that produce the polypeptide or the purification method that DNA sequence of ALT (e.g., the DNA sequence of KEGG/ will be described later. However, as long as the resultant ENZYME: 2.6.1.2/Homo sapiens (human): 2875, KEGG/ polypeptide has functions equivalent to the functions of the 40 ENZYME: 2.6.1.2/Homo sapiens (human): 84706, KEGG/ ALT derived from human or the like, a DNA encoding the ENZYME: 2.6.1.2/Mus musculus (mouse): 76282, KEGG/ polypeptide can be used in the present invention. For ENZYME: 2.6.1.2/Mus musculus (mouse): 108682, KEGG/ example, when the polypeptide of the present invention is ENZYME: 2.6.1.2/Rattus norvegicus (rat): 81670, KEGG/ expressed in a prokaryote (e.g., Escherichia coli), a methion ENZYME: 2.6.1.2/Canis familiaris (dog): 609510, KEGG/ ine reside is added to the N-terminus of the initial amino acid 45 ENZYME: 2.6.1.2/Xenopus laevis (African clawed frog): sequence of the polypeptide. When the polypeptide is 444533, KEGG/ENZYME: 2.6.1.2/Drosophila melano expressed in a eukaryote (e.g., a mammalian cell), the N-ter gaster (fruitfly): Dmel CG1640, KEGG/ENZYME: 2.6.1.2/ minal signal sequence is removed. A DNA encoding Such a Caenorhabditis elegans (nematode): C32F10.8, KEGG/EN polypeptide can be used in the present invention. ZYME: 2.6.1.2/Oryza sativa japonica (Japanese rice): In the present invention, as a DNA encoding ALT, a DNA 50 4342210, KEGG/ENZYME: 2.6.1.2/Oryza sativa japonica having the nucleotide sequence of KEGG/ENZYME: (Japanese rice): 4348524, KEGG/ENZYME: 2.6.1.2/Cyan 2.6.1.2/Homo sapiens (human): 2875, KEGG/ENZYME: idioschyzon merolae: CMM066C, KEGG/ENZYME: 2.6.1.2/Homo sapiens (human): 84706, KEGG/ENZYME: 2.6.1.2/Saccharomyces cerevisiae: YLR089c, KEGG/EN 2.6.1.2/Mus musculus (mouse): 76282, KEGG/ENZYME: ZYME: 2.6.1.2/Saccharomyces cerevisiae: YDR111c, 2.6.1.2/Mus musculus (mouse): 108682, KEGG/ENZYME: 55 KEGG/ENZYME: 2.6.1.2/Ashbya gossypii (Eremothecium 2.6.1.2/Rattus norvegicus (rat): 81670, KEGG/ENZYME: gossypii): AGOS AGR085W, KEGG/ENZYME: 2.6.1.2/ 2.6.1.2/Canis familiaris (dog): 609510, KEGG/ENZYME: Candida albicans: CaO19 346, KEGG/ENZYME: 2.6.1.2/ 2.6.1.2/Xenopus laevis (African clawed frog): 444533, Schizosaccharomyces pombe: SPBC582.08, KEGG/EN KEGG/ENZYME: 2.6.1.2/Drosophila melanogaster (fruit ZYME: 2.6.1.2/Aspergillus nidulans: AN1923.2, KEGG/ fly): Dmel CG1640, KEGG/ENZYME: 2.6.1.2/Caenorhab 60 ENZYME: 2.6.1.2/Aspergillus filmigatus: AFUA ditis elegans (nematode): C32F10.8, KEGG/ENZYME: 6G07770, KEGG/ENZYME: 2.6.1.2/Aspergillus oryzae: 2.6.1.2/Oryza sativa japonica (Japanese rice): 4342210, AO090003000164, KEGG/ENZYME: 2.6.1.2/Cryptococcus KEGG/ENZYME: 2.6.1.2/Oryza sativa japonica (Japanese neoformans JEC21: CNG01490, KEGG/ENZYME: 2.6.1.2/ rice): 4348524, KEGG/ENZYME: 2.6.1.2/Cyanidioschyzon Dictyostelium discoideum: DDB 0232139, KEGG/EN merolae: CMMO66C, KEGG/ENZYME: 2.6.1.2/Saccharo 65 ZYME: 2.6.1.2/Trypanosoma brucei: Tb927.1.3950, KEGG/ myces cerevisiae:YLR089c, KEGG/ENZYME: 2.6.1.2/Sac ENZYME: 2.6.1.2/Leishmania major: LimjF12.0630, charomyces cerevisiae: YDR111c, KEGG/ENZYME: KEGG/ENZYME: 2.6.1.2/Entamoeba histolytica. US 9,068,212 B2 29 30 233.t.00009, KEGG/ENZYME: 2.6.1.2/Entamoeba his From the resultant mRNA, cDNA is synthesized using a tolytica: 24.t00016, KEGG/ENZYME: 2.6.1.2/Trypano reverse transcriptase. Alternatively, cDNA may be synthe soma Cruzi: 506529.420, KEGG/ENZYME: 2.6.1.2/Trypa sized using a kit such as AMV Reverse Transcriptase First nosoma cruzi: 506529.430, KEGG/ENZYME: 2.6.1.2/ Strand cDNA Synthesis Kit (SEIKAGAKU CORPORA Trypanosoma cruzi: 510889. 120 or KEGG/ENZYME: TION). It is also possible to synthesize and amplify cDNA 2.6.1.2/Trypanosoma cruzi: 510889.140) as a probe. The according to the 5'-RACE method (Frohman, M. A. et al., cDNA library may be prepared, for example, by the method Proc. Natl. Acad. Sci. USA (1988) 85, 8998-9002; Belyay described in Sambrook, J. et al., Molecular Cloning, Cold sky, A. et al., Nucleic Acids Res. (1989) 17, 2919-2932) using Spring Harbor Laboratory Press (1989). Alternatively, a com 5'-Ampli FINDER RACE Kit (Clontech) and polymerase mercial clNA library may be used. It is also possible to 10 prepare the DNA encoding ALT by preparing RNA from a chain reaction (PCR) with primers. cell expressing ALT, Synthesizing oligo DNA molecules A DNA fragment of interest is prepared from the resultant based on the DNA sequence of ALT (e.g., the DNA sequence PCR product and ligated to a vector DNA to thereby prepare of KEGG/ENZYME: 2.6.1.2/Homo sapiens (human): 2875, a recombinant vector. The vector is introduced into a host KEGG/ENZYME: 2.6.1.2/Homo sapiens (human): 84706, 15 (e.g., E. coli), followed by selection of resultant colonies to KEGG/ENZYME: 2.6.1.2/Mus musculus (mouse): 76282, thereby obtain a desired recombinant vector. The nucleotide KEGG/ENZYME: 2.6.1.2/Mus musculus (mouse): 108682, sequence of the DNA of interest may be confirmed by a KEGG/ENZYME: 2.6.1.2/Rattus norvegicus (rat): 81670, known method Such as the dideoxynucleotide chain termina KEGG/ENZYME: 2.6.1.2/Canis familiaris (dog): 609510, tion method. KEGG/ENZYME: 2.6.1.2/Xenopus laevis (African clawed Further, a nucleotide sequence of higher expression effi frog): 444533, KEGG/ENZYME: 2.6.1.2/Drosophila mela ciency can be designed for the DNA encoding ALT by con nogaster (fruit fly): Dmel CG1640, KEGG/ENZYME: sidering the frequency of codon usage in the host to be used 2.6.1.2/Caenorhabditis elegans (nematode): C32F10.8, for expression (Grantham, R. et al., Nucleic Acids Research KEGG/ENZYME: 2.6.1.2/Oryza sativa japonica (Japanese (1981) 9, p. 43-74). Further, the DNA encoding ALT can be rice): 4342210, KEGG/ENZYME: 2.6.1.2/Oryza sativa 25 modified using commercially available kits or known meth japonica (Japanese rice): 4348524, KEGG/ENZYME: ods. Examples of Such modifications include, but are not 2.6.1.2/Cyanidioschyzon merolae: CMM066C, KEGG/EN limited to, digestion with restriction enzymes, insertion of ZYME: 2.6.1.2/Saccharomyces cerevisiae: YLR089c, synthetic oligonucleotides or appropriate DNA fragments, KEGG/ENZYME: 2.6.1.2/Saccharomyces cerevisiae: addition of linkers, and insertion of an initiation codon (ATG) YDR111c, KEGG/ENZYME: 2.6.1.2/Ashbya gossypii (Er 30 emothecium gossypii): AGOS AGR085W, KEGG/EN and/or a termination codon (TAA, TGA or TAG). ZYME: 2.6.1.2/Candida albicans: CaO19 346, KEGG/EN The DNA encoding ALT also includes a DNA which ZYME: 2.6.1.2/Schizosaccharomyces pombe: SPBC582.08, hybridizes to a DNA complementary to a DNA having the KEGG/ENZYME: 2.6.1.2/Aspergillus nidulans: AN1923.2, nucleotide sequence of KEGG/ENZYME: 2.6.1.2/Homo KEGG/ENZYME: 2.6.1.2/Aspergillus fumigatus: AFUA 35 sapiens (human): 2875, KEGG/ENZYME: 2.6.1.2/Homo 6G07770, KEGG/ENZYME: 2.6.1.2/Aspergillus oryzae: sapiens (human): 84706, KEGG/ENZYME: 2.6.1.2/Mus AO090003000164, KEGG/ENZYME: 2.6.1.2/Cryptococcus musculus (mouse): 76282, KEGG/ENZYME: 2.6.1.2/Mus neoformans JEC21: CNG01490, KEGG/ENZYME: 2.6.1.2/ musculus (mouse): 108682, KEGG/ENZYME: 2.6.1.2/Rat Dictyostelium discoideum: DDB 0232139, KEGG/EN tus norvegicus (rat): 81670, KEGG/ENZYME: 2.6.1.2/Canis ZYME: 2.6.1.2/Trypanosoma brucei: Tb927.1.3950, KEGG/ 40 familiaris (dog): 609510, KEGG/ENZYME: 2.6.1.2/Xeno ENZYME: 2.6.1.2/Leishmania major: LimjF12.0630, pus laevis (African clawed frog): 444533, KEGG/ENZYME: KEGG/ENZYME: 2.6.1.2/Entamoeba histolytica. 2.6.1.2/Drosophila melanogaster (fruit fly): Dmel CG1640. 233.t.00009, KEGG/ENZYME: 2.6.1.2/Entamoeba his KEGG/ENZYME: 2.6.1.2/Caenorhabditis elegans (nema tolytica: 24.t00016, KEGG/ENZYME: 2.6.1.2/Trypano tode): C32F10.8, KEGG/ENZYME: 2.6.1.2/Oryza sativa soma Cruzi: 506529.420, KEGG/ENZYME: 2.6.1.2/Trypa 45 japonica (Japanese rice): 4342210, KEGG/ENZYME: nosoma cruzi: 506529.430, KEGG/ENZYME: 2.6.1.2/ 2.6.1.2/Oryza sativa japonica (Japanese rice): 4348524, Trypanosoma cruzi: 510889. 120 or KEGG/ENZYME: KEGG/ENZYME: 2.6.1.2/Cyanidioschyzon merolae: 2.6.1.2/Trypanosoma Cruzi: 510889.140), and performing CMM066C, KEGG/ENZYME: 2.6.1.2/Saccharomyces cer PCR using the oligo DNA molecules as primers to thereby evisiae: YLR089c, KEGG/ENZYME: 2.6.1.2/Saccharomy amplify a cDNA encoding ALT. 50 ces cerevisiae:YDR111c, KEGG/ENZYME: 2.6.1.2/Ashbya Further, by determining the nucleotide sequence of the gossypii (Eremothecium gossypii): AGOS AGR085W. resultant clNA, it is possible to determine the translation KEGG/ENZYME: 2.6.1.2/Candida albicans: CaO19346, region encoding ALT and to obtain the amino acid sequence KEGG/ENZYME: 2.6.1.2/Schizosaccharomyces pombe: of ALT. Further, by Screening a genomic library using the SPBC582.08, KEGG/ENZYME: 2.6.1.2/Aspergillus nidu resultant cDNA as a probe, it is possible to isolate a genomic 55 lans: AN1923.2, KEGG/ENZYME: 2.6.1.2/Aspergillus DNA. fumigatus: AFUA 6G07770, KEGG/ENZYME: 2.6.1.2/As Specifically, the following procedures may be used. First, pergillus oryzae: AO090003000164, KEGG/ENZYME: mRNA is isolated from cells, tissues or the like expressing 2.6.1.2/Cryptococcus neoformans JEC21: CNGO1490, ALT. For the isolation of mRNA, the total RNA is prepared by KEGG/ENZYME: 2.6.1.2/Dictyostelium discoideum: known methods, for example, the guanidine ultracentrifuga 60 DDB 0232139, KEGG/ENZYME: 2.6.1.2/Trypanosoma tion method (Chirgwin, J. M. et al., Biochemistry (1979) 18, brucei: Tb927.1.3950, KEGG/ENZYME: 2.6.1.2/Leishma 5294-5299), the AGPC method (Chomczynski, P. and Sacchi, nia major: LimjF12.0630, KEGG/ENZYME: 2.6.1.2/Entam N., Anal. Biochem. (1987) 162, 156-159) or the like, and then oeba histolytica. 233.t00009, KEGG/ENZYME: 2.6.1.2/En mRNA is purified from the total RNA using mRNA Purifica tamoeba histolytica. 24.t00016, KEGG/ENZYME: 2.6.1.2/ tion Kit (Pharmacia), etc. Alternatively, mRNA may be pre 65 Trypanosoma cruzi: 506529.420, KEGG/ENZYME: 2.6.1.2/ pared directly using QuickPrep mRNA Purification Kit Trypanosoma cruzi: 506529.430, KEGG/ENZYME: 2.6.1.2/ (Pharmacia). Trypanosoma cruzi: 510889.120 or KEGG/ENZYME: US 9,068,212 B2 31 32 2.6.1.2/Trypanosoma Cruzi: 510889.140 under stringent con these ranges and may be appropriately selected depending on ditions and encodes a polypeptide functionally equivalent to the type of the cell to be cultured, the type of the desired ALT. polypeptide, and so on. Stringent conditions can be appropriately selected by those In addition to these components, trace metal elements, skilled in the art, including, for example, low stringent con Surfactants, growth cofactors, nucleosides, and the like may ditions. Low Stringent conditions refer to, for example, 42 be added. C., 2xSSC and 0.1% SDS, preferably 50° C., 2xSSC and Specific examples of Such components include amino 0.1% SDS. More preferably, high stringent conditions may be acids, such as L-alanine, L-arginine, L-asparagine, L-aspartic selected. High stringent conditions refer to, for example, 65° acid, L-cysteine, L-cystine, L-glutamine, L-glutamic acid, C., 2xSSC and 0.1% SDS. Under these conditions, as the 10 glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-me hybridization temperature is elevated, DNAs with a higher thionine, L-ornithine, L-phenylalanine, L-proline, L-serine, homology can be obtained. The above-described DNA which L-threonine, L-tryptophan, L-tyrosine, and L-valine, prefer hybridizes is preferably a DNA derived from nature, e.g., ably, L-alanine, L-arginine, L-asparagine, L-aspartic acid, cDNA or chromosomal DNA. 15 L-cystine, L-glutamine, L-glutamic acid, glycine, L-histi These DNAs isolated by hybridization techniques usually dine, L-isoleucine, L-leucine, L-lysine, L-methionine, have a high nucleotide sequence identity with a DNA encod L-phenylalanine, L-proline, L-serine, L-threonine, L-tryp ing the ALT derived from human or the like. The DNA encod tophan, L-tyrosine and L-Valine; Vitamins, such as i-inositol, ing ALT also includes a DNA which encodes a polypeptide biotin, folic acid, lipoic acid, nicotinamide, nicotinic acid, functionally equivalent to the ALT derived from human or the p-aminobenzoic acid, calcium pantothenate, pyridoxal like and has high identity with a DNA encoding the ALT hydrochloride, pyridoxine hydrochloride, riboflavin, thia derived from human or the like. The term “high identity” mine hydrochloride, vitamin B and ascorbic acid, prefer refers to usually 96% or more homology, preferably 98% or ably, biotin, folic acid, lipoic acid, nicotinamide, calcium more homology, more preferably 99% or more identity. The pantothenate, pyridoxal hydrochloride, riboflavin, thiamine identity of nucleotide sequences may be determined by algo 25 hydrochloride, Vitamin B and ascorbic acid; lipid factors, rithm BLAST (Karlin and Altschul, Proc. Natl. Acad. Sci. Such as choline chloride, choline tartrate, linoleic acid, oleic USA 90:5873-5877, 1993). Based on this algorithm, pro acid and cholesterol, preferably, choline chloride; energy grams such as BLASTN and BLASTX have been developed Sources. Such as glucose, galactose, mannose, and fructose, (Altschul et al. J. Mol. Biol. 215:403-410, 1990). When preferably, glucose; osmotic regulators, such as sodium chlo nucleotide sequences are analyzed by BLASTN based on 30 ride, potassium chloride, and potassium nitrate, preferably, BLAST, parameters may be set as score=100 and wordlength=12, for example. Specific procedures for these sodium chloride; iron sources, such as iron EDTA, ferric analysis methods are known (ncbi.nlm.nih.gov.). citrate, ferrous chloride, ferric chloride, ferrous sulfate, ferric Production of a desired polypeptide may be performed by sulfate, and ferric nitrate, preferably, ferric chloride, iron transferring a gene encoding the desired polypeptide into a 35 EDTA, and ferric citrate; and pH regulators, such as Sodium cell which strongly expresses a bicarbonate transporter and hydrogencarbonate, calcium chloride, Sodium dihydrogen CSAD or ALT and culturing the resultant cell in a medium. phosphate, HEPES and MOPS, preferably, sodium hydro When a desired polypeptide is produced using a cell into gencarbonate. Culture media containing any of these compo which a bicarbonate transporter gene and a CSAD or ALT nents may be given as examples. gene have been artificially transferred, the order of the trans 40 Besides the above components, there may be added trace fer of a bicarbonate transporter gene, the transfer of a CSAD metal elements, such as copper Sulfate, manganese Sulfate, or gene and the transfer of a gene encoding a desired polypep Zinc sulfate, magnesium Sulfate, nickel chloride, tin chloride, tide is not particularly limited. A gene encoding a desired magnesium chloride and sodium Subsilicate, preferably, cop polypeptide may be transferred after the transfer of a bicar per Sulfate, Zinc sulfate and magnesium sulfate; surfactants, bonate transporter gene and a CSAD or ALT gene. Alterna 45 such as Tween 80 and Pluronic F68; growth cofactors, such as tively, a bicarbonate transporter gene and a CSAD or ALT recombinant insulin, recombinant IGF-1, recombinant EGF, gene may be transferred after the transfer of a gene encoding recombinant FGF, recombinant PDGF, recombinant TGF-C., a desired polypeptide. It is also possible to transfer a bicar ethanolamine hydrochloride, Sodium selenite, retinoic acid bonate transporter gene, a CSAD or ALT gene and a gene and putrescine dihydrochloride, preferably, Sodium selenite, encoding a desired polypeptide simultaneously. 50 ethanolamine hydrochloride, recombinant IGF-1 and A bicarbonate transporter gene, a CSAD or ALT gene and putrescine dihydrochloride; and nucleosides, such as deoxy a gene encoding a desired polypeptide may be transferred adenosine, deoxycytidine, deoxyguanosine, adenosine, cyti simultaneously in a single vector. Alternatively, they may be dine, guanosine and uridine. In preferable examples of above transferred separately using a plurality of vectors. media, antibiotics, such as streptomycin, penicillin-G potas For culturing the cell which strongly expresses a bicarbon 55 sium and gentamicin, and pH-indicators, such as Phenol Red, ate transporter (and which may strongly express CSAD or may be contained. ALT), media used in conventional cell culture (preferably, The pH of the medium varies depending on the cell to be animal cell culture) may be used. These media usually con cultured. Generally, pH 6.8-7.6 is appropriate. In many cases, tain amino acids, vitamins, lipid factors, energy sources, pH 7.0–7.4 is appropriate. osmotic regulators, iron Sources and pH regulators. The con 60 It is also possible to use a commercial medium for animal tents of these components are usually as follows: amino acids cell culture, e.g., D-MEM (Dulbecco's Modified Eagle 0.05-1500 mg/L, vitamins 0.001-10 mg/L, lipid factors 0-200 Medium), D-MEM/F-12 1:1 Mixture (Dulbecco's Modified mg/L, energy sources 1-20 g/L, osmotic regulators 0.1-10000 Eagle Medium Nutrient Mixture F-12), RPMI1640, CHO-S- mg/L, iron sources 0.1-500 mg/L, pH regulators 1-10000 SFMII (Invitrogen), CHO-SF (Sigma-Aldrich), EX-CELL mg/L, trace metal elements 0.00001-200 mg/L, surfactants 65 301 (JRH Biosciences), CD-CHO (Invitrogen), IS CHO-V 0-5000 mg/L, growth cofactors 0.05-10000 ug/L and nucleo (Irvine Scientific), PF-ACF-CHO (Sigma-Aldrich) or the sides 0.001-50 mg/L. However, the contents are not limited to like. US 9,068,212 B2 33 34 Alternatively, the medium may be a serum-free medium. neal injection, Subcutaneous injection, etc.), transnasal When the cell which strongly expresses a bicarbonate administration, transpulmonary administration, transdermal transporter (and which may strongly express CSAD or ALT) administration and the like may be enumerated. is CHO cells, CHO cells may be cultured by methods known The present invention provides a cell which has a trans to those skilled in the art. For example, CHO cells may be ferred DNA encoding a bicarbonate transporter and a trans cultured usually in an atmosphere with a CO concentration ferred DNA encoding cysteine sulfinic acid decarboxylase or in the gas phase of 0 to 40%, preferably 2 to 10%, at 30 to 39° alanine aminotransferase, both or either of which may be C., preferably about 37° C. incorporated into a vector. An appropriate culture period for producing a desired When eukaryotes are used, animal cells, plant cells, fungal polypeptide using the cell which strongly expresses a bicar 10 cells, etc. may be used as the host. Specific examples of bonate transporter (and which may strongly express CSAD or animal cells include mammalian cells, such as CHO cells (J. ALT) is usually 1 day to 3 months, preferably 1 day to 2 Exp. Med. (1995) 108,945), COS cells, 3T3 cells, myeloma months, more preferably 1 day to 1 month. cells, BIM (baby hamster kidney) cells, HeLa cells and Vero With respect to various culture devices for animal cell cells; amphibian cells, such as oocytes of Xenopus laevis culture, a fermentor type tank culture device, an airlift type 15 (Valle, et al., Nature (1981) 291, 358-340); or insect cells, culture device, a culture flask type culture device, a spinner such as Sf9. Sf21 and Tn5 cells. Among CHO cells, dhfr-CHO flask type culture device, a microcarrier type culture device, a lacking DHFR gene (Proc. Natl. Acad. Sci. USA (1980) 77. fluidized bed type culture device, a hollow fiber type culture 4216-4420) and CHO K-1 (Proc. Natl. Acad. Sci. USA device, a roller bottle type culture device, a packed bed type (1968) 60, 1275) are used with particular advantage. When culture device, or the like may be used. high expression is intended in an animal cell, CHO cells are Culture may be performed by any culture method such as especially preferred. Introduction of the DNA which may be batch culture, fed-batch culture or continuous culture. Pref incorporated into a vector into the host cell may be performed erably, fed-batch culture or continuous culture is used. Fed by such methods as the calcium phosphate method, the DEAE batch culture is more preferred. dextran method, a method using a cationic ribosome DOTAP When the polypeptide produced according to the method 25 (Boehringer-Mannheim), electroporation, lipofection, etc. of the present invention has a biological activity useful as a As plant cells for polypeptide production, a Nicotiana pharmaceutical, it is possible to produce a pharmaceutical by tabacum-derived cell is known as a polypeptide production mixing this polypeptide with pharmaceutically acceptable system and this may be subjected to callus culture. As fungal carriers or additives and formulating into a preparation. cells for polypeptide production, specific examples include Specific examples of pharmaceutically acceptable carriers 30 yeast belonging to the genus Saccharomyces, e.g., Saccharo and additives include water, organic solvents that are phar myces cerevisiae, and filamentous fungi belonging to the maceutically acceptable, collagen, polyvinyl alcohol, polyvi genus Aspergillus, e.g., Aspergillus niger: nylpyrrolidone, carboxyvinyl polymer, carboxymethylcellu When prokaryotes are used, production systems using bac lose Sodium, Sodium polyacrylate, Sodium alginate, water terial cells are known. Specific examples of such bacterial soluble dextran, carboxymethyl Starch sodium, pectin, 35 cells include E. coli (such as JM109, DH5C, HB101) and methylcellulose, ethyl cellulose, Xanthan gum, gum Arabic, Bacillus subtilis. casein, agar-agar, polyethylene glycol, diglycerin, glycerin, The polypeptide encoded by a gene of interest may be propylene glycol, petrolatum, paraffin, Stearyl alcohol, obtained by transforming these cells with the gene of interest Stearic acid, human serum albumin (HSA), mannitol, Sorbi and culturing the transformed cells in vitro. The culture may tol, lactose, and Surfactants that are acceptable as pharmaceu 40 be performed by known methods. For example, as a culture tical additives. broth for animal cells, a medium such as DMEM, MEM, Actual additives may be selected from the above-men RPMI1640 or IMDM may be used. A serum supplement such tioned additives singly or in combination according to the as fetal calf serum (FCS) may be used jointly. Alternatively, dosage form of the therapeutic of the present invention, but serum-free culture may be performed. The pH during culture are not limited to those listed above. For example, when a 45 is preferably about 6 to 8. The culture is usually performed at polypeptide is used in an injectable formulation, the purified about 30-40°C. for about 15-200 hours. If necessary, replace polypeptide may be dissolved in a solvent such as physiologi ment of the medium, aeration and agitation are carried out. cal saline, buffer or a glucose solution, and then an adsorption On the other hand, in vivo production systems include inhibitor such as Tween 80, Tween 20, gelatin or human those using animals or plants. A gene of interest is transferred serum albumin may be added to the solution. Alternatively, a 50 into these animals or plants to produce the polypeptide in the freeze-dried agent may be used to prepare a dosage form animal bodies or plant bodies. Then, the polypeptide is col which is dissolved and reconstituted prior to use. Examples of lected. The term "host’ as used herein includes such animals the excipient useful for freeze-drying include Sugar alcohols or plants. and saccharides such as mannitol and glucose. When animals are used, available production systems Effective doses of the polypeptide may be appropriately 55 include those using mammals or insects. Goat, pig, sheep, selected depending on the type of the polypeptide, the type of mouse and cattle may be used as mammals (Vicki Glaser, the disease to be treated or prevented, the age of the patient, SPECTRUM Biotechnology Applications, 1993). When the severity of the disease, etc. For example, when the mammals are used, transgenic animals may be used. polypeptide is anti-glypican antibody, the effective dose of First, a gene of interest is fused to a gene encoding a anti-glypican antibody is selected within a range of 0.001 mg 60 polypeptide produced inherently in milk (such as goat to 1000 mg per kg of body weight per administration. Alter (B-casein) to thereby prepare a fusion gene. A DNA fragment natively, a dose of 0.01-100000 mg/body may be selected per containing this fusion gene is injected into a goat embryo, patient. However, effective dose is not limited to these ranges. which is then implanted in the uterus of a female goat. The The polypeptide may be administered either orally or polypeptide of interest can be obtained from the milk pro parenterally, but parenteral administration is preferred. Spe 65 duced by transgenic goats born from the goat which accepted cifically, injection (e.g., systemic or local administration by the embryo or the offspring of the transgenic goats. In order to intravenous injection, intramuscular injection, intraperito increase the yield of milk containing the polypeptide pro US 9,068,212 B2 35 36 duced by the transgenic goats, hormones may be appropri Example 1 ately administered to the transgenic goats (Ebert, K. M. et al., Bio/Technology (1994) 12, 699-702). Cloning of a Human Hepatic Cell Anion Exchanger Examples of insects which may be used include silkworm. (Anion Exchanger 1, Band 3) Gene In this case, silkworm is infected with baculovirus carrying a 5 transferred gene encoding the polypeptide of interest. The Using a commercial Human Liver QUICK-Clone cDNA polypeptide of interest can be obtained from the body fluid of (Clontech Laboratories, Inc.) as a template, an Anion the silkworm (Susumu, M. et al., Nature (1985) 315, 592 Exchanger (AE 1) gene derived from a human liver was 594). obtained by a PCR method. The gene thus cloned was Furthermore, when plants are used, tobacco can typically 10 sequenced to confirm that it encoded AE1 in view of its be used. When tobacco is used, a gene encoding the polypep homology with a published human AE1. The AE1 gene thus obtained had mutations at eight sites in the sequence of 2733 tide of interest is inserted into a plant expression vector (e.g., bases (t263g, t357c. a645t, ag72c, c951t, a2078g, t2195c, pMON 530), which is then transferred into a bacterium such c2500t) and coded for 911 amino acids including four differ as Agrobacterium tumefaciens. A tobacco plant (e.g., Nicoti 15 ent amino acids (L88R, E693G, V732A, H834Y). However, ana tabacum) is infected with the resultant bacterium. The because a product obtained by the gene was predicted to be a polypeptide of interest can be obtained from leaves of this transporter having 13 transmembrane domains (FIG. 1), the plant (Julian, K.-C. Ma et al., Eur. J. Immunol. (1994) 24, gene was used for cell modulation as an AE1 gene derived 131-138). from a human liver. The polypeptide thus obtained can be isolated from the inside of the host cell or from its outside (e.g., medium), and Example 2 purified to a Substantially pure and homogeneous polypep tide. Isolation and purification of polypeptides can be per Increase in the Amount of Antibody Production by formed using conventional isolation and purification methods Introduction of a Human Anion Exchanger Gene for polypeptides, and are not limited in any way. For example, 25 polypeptides can be isolated and purified by appropriate By adding a Kozak sequence to the human AE1 gene selection and combination of various tools and techniques, obtained by PCR cloning in Example 1 (which is hereinafter Such as chromatography columns, filters, ultrafiltration, salt called AE1), pHyg-AE1 (FIG.2) and pPur-AE1 (FIG.3) were ing-out, precipitation with solvent, extraction with solvent, constructed as CMV promoter expression plasmids. The distillation, immunoprecipitation, SDS-polyacrylamide gel 30 pHyg-AE1 or pHyg expression plasmids that did not contain electrophoresis, isoelectric focusing, dialysis, recrystalliza the AE1 gene (which was obtained by first introducing tion, etc. Hygromycin-resistance gene expression units derived from Examples of chromatography include affinity chromatog pTK5 provided by Clontech Laboratories, Inc. into pSV2 raphy, ion exchange chromatography, hydrophobic chroma dhfr plasmids (ATCC No. 37146) and then removing the dhfr 35 expression units from the constructed plasmids) were intro tography, gel filtration, reverse-phase chromatography, duced into anti-glypican-3 antibody-producing CHO cells as adsorption chromatography, etc. (Strategies for Protein Puri a parent strain (see International Publication WO 2006/ fication and Characterization: A Laboratory Course Manual. 006693) by electroporation. Then, strains that exhibited high Ed. Daniel R. Marshak et al., Cold Spring Harbor Laboratory proliferation in static culture in the presence of Hygromycin Press, 1996). These chromatographic techniques can be car 40 (200 ug/ml) were selected. After amplification, a total RNA ried out using liquid phase chromatography, for example, was prepared from the pHyg-AE1 strains, and five strains HPLC, FPLC, etc. The present invention also includes those expressing human AE1 at high levels were selected by a polypeptides highly purified using these purification meth Taq Man method. Further, a comparison was made for the ods. amount of antibody production between pHyg-transformed Before or after the purification, it is also possible to give 45 cells as a control (four strains) and four strains of human optional modifications to the polypeptide or remove a partial AE1-transformed cells that proliferated at a level equivalent peptide therefrom by reacting the polypeptide with an appro to that observed with control during shake culture. During priate polypeptide modification enzyme. Examples of Such fed-batch culture in a 50-ml shaker flask under the condition enzyme include, but are not limited to, trypsin, chymotrypsin, of 2x10 cells/mL in an initial stage, the amount of an anti lysyl endopeptidase, protein kinase and glucosidase. 50 glypican-3 antibody produced by pHyg-AE1-transformed In the present invention, the concept of “cells into which cells (four strains) on day 12 after initiation of the shake DNA has been transferred encompasses not only cells into culture was significantly greater than that produced by pHyg which exogenous DNA has been incorporated by genetic transformed cells (four strains) (t-test: P-0.05, FIG. 4). recombination technology; but also cells in which endog Then, using as a parent strain the AE1 expressing strain that enous DNA has been activated by gene activation technology 55 produced the largest amount of an antibody among the four (see, for example, International Publication WO94/12650) so pHyg-AE1-transformed strains, pPur-CSAD or a cysteine that expression of a protein corresponding to the endogenous sulfinic acid decarboxylase (CSAD) expression plasmid con DNA or transcription of the DNA has been initiated or taining a Puromycin-resistance gene (FIG.10, see Referential increased. Example 2 described later), pPur-ALT1 or an alanine ami 60 notransferase (ALT1) expression plasmid containing Puro EXAMPLES mycin-resistance gene (FIG. 11, see Referential Example 4 described later), and a control plasmid pPur (pPUR, Puromy Hereinbelow, the present invention will be described in cin resistance expression vector, provided by Clontech Labo more detail with reference to the following Examples. It ratories, Inc.) were introduced by electroporation. Then, should be noted that these Examples are provided only for 65 strains that exhibited high proliferation in static culture in the illustrating the present invention and not for limiting the presence of Puromycin (6 ug/ml) were selected. After ampli Scope of the present invention. fication, a total RNA was prepared from the strains thus US 9,068,212 B2 37 38 selected. Then, AE1/CSAD co-expressing strains (nine anti-IL-6 receptor antibody gene had been transferred) (Japa strains), AE1/ALT1 co-expressing strains (10 Strains), and nese Unexamined Patent Publication No. Hei 8-99902), and AE1/pPur co-expressing strains (eight strains), which then cDNA was synthesized therefrom in a poly(A) depen expressed the newly introduced genes at high levels, were dent manner. Hamster CSAD and CDO1 genes were obtained selected and compared for the amount of antibody production 5 and the survival rate. In fed-batch culture in 50-mL shaker by PCR using as a template the cDNA fragmented with three flasks under the condition of 2x10 cells/mL in an initial restriction enzymes, SalI, XhoI and EcoRI. As PCR primers, stage, the AE1/CSAD co-expressing strains (nine strains) those containing the 5'-end and the 3'-end sequence con showed significantly greater amounts of anti-glypican-3 anti served between rat and mouse CSADs or CDO1s were body production (t-test PK0.05, FIG. 5) and significantly designed. The nucleotide sequences of the cloned genes were higher survival rates (t-test P-0.01, FIG. 6) than the control 10 determined. From its homology with other CSAD or CDO1 AE1/pPurco-expressing strain (eight strains) on day 10 at the genes of known species, the cloned gene was confirmed to late stage of the shake culture. Among the three kinds of encode hamster CASD (FIG.9). The amino acid sequence of co-expressing strains, AE1/ALT1 co-expressing strains (10 hamster CSAD has high homology with the known amino strains) produced the largest amount of an anti-glypican-3 acid sequences of mouse CSAD (96% identity), rat CSAD antibody, which was significantly greater than that produced 15 (96% identity) and human CSAD (91% identity); it was pre by the control AE1/ppur co-expressing strains (eight Strains) dicted that hamster CSAD is an enzyme having the same on day 8 of the shaker fed-batch culture (t-test P-0.01, FIG. activity. The nucleotide sequence of hamster CSAD is shown 7). Subsequently, AA53, which produced the largest amount in SEQID NO:3. The amino acid sequence of hamster CSAD of an antibody (1497 mg/L/8 days) and expressed ALT1 mRNA at the highest level among the AE1/ALT1 co-express is shown in SEQID NO: 4. ing strains (10 Strains) in the study using the shaker fed-batch Referential Example 2 culture, was subjected to fed-batch culture in a 1-Ljar (10x 10 cells/mL in an initial stage). Then, the amount of an antibody produced by AA53 on day 7 of the culture was found Construction of a Hamster CSAD Expressing to be 1.9 g/L/7 days, revealing that AA53 was capable of Plasmid for Puromycin-Selection high-yield antibody production in short-term culture (FIG. 8). 25 Considering that TA41, which was a TauT/ALT1 co-express By adding a Kozak sequence to the hamster CSAD (which ing strain that produced 5.3 g/L of an antibody on day 21 of is hereinafter called CSAD) gene obtained by PCR cloning in the culture (see Referential Example 4 described later), pro Referential Example 1, a CMV promoter expression plasmid duced 1.5 g/L of an antibody on day 7 of the culture. AA53 has pPur/CSAD (FIG. 10) was constructed. a potential to produce a greater amount of an antibody in a short time than does TA41, and hence, AA53 is considered to Referential Example 3 be suitable for practical production. The above results show that cells capable of high-yield Cloning of Human Hepatic Cell Alanine antibody production can be obtained by strongly expressing Aminotransferase Gene an anion exchanger (AE 1) artificially, and by strongly 35 expressing AE1 and CSAD or ALT1 simultaneously. Using a commercial Human Liver QUICK-Clone cDNA Also, the effect of strongly expressing AE1 was shown by (Clontech Laboratories, Inc.) as a template, alanine ami construction of a strain capable of producing an anti-IL-6R notransferase (ALT1) gene derived from a human liver was antibody using an AE1 strongly expressing host cell. Into an obtained by a PCR method. The gene thus cloned was ordinary host cell DXB11, pHyg-AE1 (FIG. 2) was intro 40 sequenced and confirmed to encode ALT1 based on its duced by electroporation. Then, strains that exhibited high homology with published human ALT1. The ALT1 gene thus proliferation in static culture in the presence of Hygromycin obtained had mutations at five sites in the sequence of 1488 (200 ug/ml) were selected. After amplification, cells express bases (c157a, a215g, c765t, t857c, t995a) and coded for 496 ing human AE1 at high levels were established as an AE1/ DXB11 host cell by a TaqMan method. Anti IL-6R antibody amino acids including four different amino acids (R53S, expression plasmids were introduced into the AE1/DXB11 45 Q72R, F286S, M332K), but this was used as a PCR clone of host cells, and AE1-S08 was obtained by single cell cloning. the human liver derived ALT1 for cell modulation. AE1-S08 thus obtained was capable of high-yield production of an anti-IL-6R antibody, and the amount of its production Referential Example 4 on day 14 offed-batch culture in a 1 L-jar (7x10 cells/mL in an initial stage) was 3.0 g/L as shown in FIG. 12. It has been Increase in Antibody Yield by Transfer of Human confirmed that AE1-S08 capable of high-yield antibody pro Alanine Aminotransferase duction and the host cells, AE1/DXB11, were both stable in a stability test performed by subculturing and maintained AE1 By adding a Kozak sequence to the human ALT1 obtained expression at high levels. by cloning in Referential Example 3 (which is hereinafter The above results suggest that the effect of introduction of 55 called ALT1), pPur-ALT1, which was a CMV promoter an AE1 gene acts positively both before and after introduction expression plasmid, was constructed (FIG. 11). The pPur of an antibody gene. ALT1 or pPur expression plasmids that did not contain the The present invention can be applied to all types of cells ALT1 gene were introduced into anti-glypican-3 antibody capable of producing a polypeptide (preferably an antibody). producing CHO cells as parent strains (see International Pub Referential Example 1 60 lication WO 2006/006693) by electroporation, and cell strains that exhibited high proliferation in static culture in the Cloning of CHO Cell-Derived Hamster Cysteine presence of Puromycin (6 ug/ml) (pPur-ALT1: seven strains, Sulfinic Acid Decarboxylase (CSAD) and Cysteine pPur: three strains) were selected. After expansion, a total Dioxygenase, type I (CDO1) Genes RNA was prepared from the pPur-ALT1 cell strains, and six 65 strains expressing human ALT1 at high levels were selected Total RNA was extracted from anti-IL-6 receptor anti by a TaqMan method. Further, a comparison was made for the body-producing cells (ACHODMB11 cell line into which an antibody yield between pPur-transferred cells as a control US 9,068,212 B2 39 40 (three strains) and four strains of human ALT1-transferred the cloned gene was confirmed to encode hamster TauT (FIG. cells that proliferated at a level equivalent to that observed 13). The amino acid sequence of hamster TauT has high with the pPur-transferred cells during the shake culture. Dur homology with mouse TauT (96% identity), rat TauT (96% ing fed-batch culture in a 50 ml shaker flask with an initial cell identity) and human TauT (93% identity); it was predicted density of 2x10 cells/mL, the anti-glypican-3 antibody yield 5 that hamster TauT is a transporter with 12 transmembrane of pPur-ALT1-transferred cells (four strains, 1236+149 regions (FIG. 14). mg/L) on day 17 at the late stage of the shaker culture was significantly higher than that of pPur-transferred cells (three Referential Example 6 strains, 871 +119 mg/L) (t-test: p-0.01). A72, a pPur-ALT1 expressing strain, and P41, a pPur expressing strain, were 10 Increase in Viable Cell Density, Inhibition of Lactate each found to have produced the largest amount of an anti Production and Increase in Antibody Yield, as body in the study using shaker fed-batch culture, and they Caused by Transfer of Hamster Taurine Transporter were subjected to fed-batch culture in 1 Ljars (an initial cell density of 10x10 cells/mL). As a result, the antibody yield of CMV promoter expression plasmid pHyg/TauT was con A72 was 2.9 g/L on day 19 of the culture, which was greater 15 than the antibody yield of P41 (2.2 g/L). Since no increase structed (FIG. 15) by adding Kozak sequence to the hamster was observed in the antibody yield of P41 on day 14 or TauT (hereinafter, TauT) gene obtained by cloning in Refer Subsequent days after the initiation of the culture, the high ential Example 5. Control plasmid pHyg without pHyg/TauT yield production of an antibody by A72 was considered to be or TauT gene was introduced by electroporation into the attributable to the survival ratio maintaining effect (The sur parent strain anti-glypican-3 antibody producing CHO cell vival rates of pPur-ALT1 expressing strain A72 and pPur (see WO 2006/006693). After selection of expression plas expressing strain P41 were 60% and 23%, respectively, on mid-transferred cells in the presence of hygromycin (400 day 14 of the culture). ug/ml), all of the stably growing cell strains were expanded Then, pPur-ALT1 or pPur was co-transferred into T10 (pHyg/TauT: 8 strains; pHyg: 7 strains). TauT mRNA was which was a pHyg-TauT-transferred cell used as a parent 25 prepared. Subsequently, 7 strains were confirmed to express strain (see Referential Example 6 described later). TauT/ TauT more strongly than the parent strain by the TaqMan ALT1 co-expressing cells that exhibited high proliferation method; they were selected as pHyg/TauT transferred cells. and expressed human ALT1 at high level (six strains) and The mean mRNA expression level of these transferred cells (7 TauT/pPur co-expressing cells that exhibited high prolifera strains) was about 40 times larger than the control (7 Strains). tion (eight strains) were selected and subjected to fed-batch 30 Cells of the total 14 strains were subjected to batch culture culture in 50 mL shaker flasks (an initial cell density of and fed-batch culture in 50 ml shaker flasks with an initial cell 10x10 cells/mL). The anti-glypican-3 antibody yield density of 2x10 cells/ml. On day 7 of culture (late-stage), (745+87 mg/L) of TauT/ALT1 co-expressing cells, which viable cell densities, lactate yields and anti-glypican-3 anti were ALT expressing cells, on day 4 of the shaker culture was body yields in those strains were compared. In batch culture, significantly higher than that of TauT/pPur cells (616+29 35 mg/L) (t-test: p-0.01). growth inhibitory Substances Such as lactate accumulate in TA41, which was a TauT/ALT1 co-expressing strain that culture broth as cells grow and their growth is inhibited. produced the largest amount of an antibody (88.1 mg/L/4 However, the viable cell densities (9.28+3.27x10 cells/ml) days) and expressed ALT1 mRNA at the highest level in the and lactate yields (1.54+0.20 g/L) in pHyg/TauT transferred study using the shaker fed-batch culture, was subjected to 40 cells were superior to those in pHyg transferred cells (viable fed-batch culture in a 1 Ljar (an initial cell density of 10x10 cell densities: 5.69+2.09x10 cells/ml, lactate yields: cells/mL). The antibody yields were as high as 1.3 g/L on day 1.54+0.20 g/L) (t test; p<0.05). With respect to anti-glypi 7 of the culture, 3.0 g/L on day 10 of the culture, 3.5 g/L on can-3 antibody yield, 4 out of the 7 strains of pHyg/TauT day 12 of the culture, 4.6 g/L on day 17 of the culture, and 5.3 transferred cell showed antibody yields (mean antibody g/L on day 21 of the culture, which were clearly higher than 45 yield: 440.6 mg/L) higher than the highest yield in pHyg the values for TP08 (656 mg/L/4 days), which was a control transferred cell (389.6 mg/L). Further, since superiority of strain that produced the largest amount of an antibody among pHyg/TauT transferred cells in anti-glypican-3 antibody the TauT/pPur co-expressing strains (2.4 g/L on day 10 of the yield became more evident (t test; P-0.01) in fed-batch cul culture). ture, pHyg/TauT transferred T10 strain (which showed the 50 highest growth ability among the above 4 strains) and the Referential Example 5 parent strain were subjected to fed-batch culture in 1 Ljar. As a result, the viable ratio of T10 was maintained at 80% or Cloning of CHO Cell-Derived Hamster Taurine more even on day 32 of culture, with inhibited lactate pro Transporter Gene duction. Consequently, its anti-glypican-3 antibody yield 55 achieved 2.9 g/L on day 35 of culture. It was confirmed by Total RNA was extracted from anti-IL-6 receptor anti flow cytometric analysis that TauT-transferred T10 cell was body-producing cells (ACHODXB11 cell line into which an expressing TauT molecules on the cell membrane. These anti-IL-6 receptor antibody gene had been transferred) (Japa results suggest that by artificially expressinghamster Taut, it nese Unexamined Patent Publication No. Hei 8-99902), and is possible to raise the potential of antibody-producing cells then cDNA was synthesized therefrom in a poly(A) depen 60 and create strains capable of enhanced antibody production. dent manner. Hamster taurine transporter (TauT) gene was All publications, patent and patent applications cited obtained by PCR using as a template the cDNA fragmented herein are incorporated herein by reference in their entirety. with three restriction enzymes, Salt XhoI and EcoRI. As PCR primers, those containing the 5'-end and the 3'-end sequence INDUSTRIAL APPLICABILITY conserved between rat and mouse TauTs were designed. The 65 nucleotide sequence of the cloned gene was determined. The present invention is applicable to production of From its homology with other TauT genes of known species, polypeptides.

US 9,068,212 B2 45 46 - Continued

His Ser His Ala Gly Glu Lell Glu Ala Luell Gly Gly Val Lys Pro Ala 1.65 17O 17s

Wall Luell Thir Arg Ser Gly Asp Pro Ser Glin Pro Lell Lell Pro Glin His 18O 185 19 O

Ser Ser Luell Glu Thir Glin Lell Phe Glu Glin Gly Asp Gly Gly Thir 195

Glu Gly His Ser Pro Ser Gly Ile Luell Glu Lys Ile Pro Pro Asp Ser 21 O 215

Glu Ala Thir Luell Wall Lell Wall Gly Arg Ala Asp Phe Lell Glu Glin Pro 225 23 O 235 24 O

Wall Luell Gly Phe Wall Arg Lell Glin Glu Ala Ala Glu Lell Glu Ala Wall 245 250 255

Glu Luell Pro Wall Pro Ile Arg Phe Luell Phe Wall Lell Lell Gly Pro Glu 26 O 265 27 O

Ala Pro His Ile Asp Thir Glin Luell Gly Arg Ala Ala Ala Thir Luell 27s 285

Met Ser Glu Arg Wall Phe Arg Ile Asp Ala Met Ala Glin Ser Arg 29 O 295 3 OO

Gly Glu Luell Luell His Ser Lell Glu Gly Phe Luell Asp Ser Luell Wall 3. OS 310 315

Lell Pro Pro Thir Asp Ala Pro Ser Glu Glin Ala Lell Lell Ser Luell Wall 3.25 330 335

Pro Wall Glin Arg Glu Lell Lell Arg Arg Arg Glin Ser Ser Pro Ala 34 O 345 35. O

Pro Asp Ser Ser Phe Lys Gly Lieu Asp Lieu Asn Gly Gly Pro 355 360 365

Asp Asp Pro Luell Glin Glin Thir Gly Glin Luell Phe Gly Gly Luell Wall Arg 37 O 375

Asp Ile Arg Arg Arg Tyr Pro Luell Ser Asp Ile Thir Asp Ala 385 390 395 4 OO

Phe Ser Pro Glin Wall Lell Ala Ala Wall Ile Phe Ile Phe Ala Ala 4 OS 415

Lell Ser Pro Ala Ile Thir Phe Gly Gly Luell Luell Gly Glu Lys Thir Arg 425 43 O

Asn Glin Met Gly Wall Ser Glu Luell Luell Ile Ser Thir Ala Wall Glin Gly 435 44 O 445

Ile Luell Phe Ala Lell Lell Gly Ala Glin Pro Luell Lell Wall Wall Gly Phe 450 45.5 460

Ser Gly Pro Luell Lell Wall Phe Glu Glu Ala Phe Phe Ser Phe Glu 465 470

Thir Asn Gly Luell Glu Tyr Ile Wall Gly Arg Wall Trp Ile Gly Phe Trp 485 490 495

Lell Ile Luell Luell Wall Wall Lell Wall Wall Ala Phe Glu Gly Ser Phe Luell SOO 505 51O

Wall Arg Phe Ile Ser Arg Thir Glin Glu Ile Phe Ser Phe Luell Ile 515 525

Ser Luell Ile Phe Ile Glu Thir Phe Ser Lell Ile Ile Phe 53 O 535 54 O

Glin Asp His Pro Lell Glin Thir Asn Tyr Asn Wall Luell Met Wall 5.45 550 555 560

Pro Pro Glin Gly Pro Lell Pro Asn Thir Ala Lell Lell Ser Luell Wall 565 st O sts

Lell Met Ala Gly Thir Phe Phe Phe Ala Met Met Lell Arg Phe US 9,068,212 B2 47 48 - Continued

585 59 O

Asn Ser Ser Phe Pro Gly Lys Luell Arg Arg Wall Ile Gly Asp Phe 595 605

Gly Wall Pro Ile Ser Ile Lell Ile Met Wall Luell Wall Asp Phe Phe Ile 610 615

Glin Asp Thir Thir Glin Luell Ser Wall Pro Asp Gly Phe Llys Val 625 630 635 64 O

Ser Asn Ser Ser Ala Arg Gly Trp Wall Ile His Pro Lell Gly Lieu. Arg 645 650 655

Ser Glu Phe Pro Ile Trp Met Met Phe Ala Ser Ala Lell Pro Ala Lieu 660 665 67 O

Lell Wall Phe Ile Lell Ile Phe Luell Glu Ser Glin Ile Thir Thir Lieu. Ile 675 685

Wall Ser Pro Glu Arg Lys Met Wall Gly Ser Gly Phe His Lieu. 69 O. 695 7 OO

Asp Luell Luell Luell Wall Wall Gly Met Gly Gly Wall Ala Ala Luell Phe Gly 7 Os 72O

Met Pro Trp Luell Ser Ala Thir Thir Wall Arg Ser Wall Thir His Ala Asn 72 73 O 73

Ala Luell Thir Wall Met Gly Ala Ser Thir Pro Gly Ala Ala Ala Glin 740 74. 7 O

Ile Glin Glu Wall Lys Glu Glin Arg Ile Ser Gly Lell Lell Wall Ala Wall 760 765

Lell Wall Gly Luell Ser Ile Lell Met Glu Pro Ile Lell Ser Arg Ile Pro 770 775

Lell Ala Wall Luell Phe Gly Ile Phe Luell Tyr Met Gly Wall Thir Ser Luell 79 O 79. 8OO

Ser Gly Ile Glin Lell Phe Asp Arg Ile Luell Luell Lell Phe Pro Pro 805 810 815

His Pro Asp Wall Pro Wall Arg Wall Thir Trp Arg 825 83 O

Met His Luell Phe Thir Gly Ile Glin Ile Ile Lell Ala Wall Lieu. Trp 835 84 O 845

Wall Wall Ser Thir Pro Ala Ser Luell Ala Luell Pro Phe Wall Lieu. Ile 850 855 860

Lell Thir Wall Pro Lell Arg Arg Wall Luell Luell Pro Lell Ile Phe Arg Asn 865 87s 88O

Wall Glu Luell Glin Cys Lell Asp Ala Asp Asp Ala Ala Thir Phe Asp 885 890 895

Glu Glu Glu Gly Arg Asp Glu Asp Glu Wall Ala Met Pro Wall 9 OO 905 91 O

SEQ ID NO 3 LENGTH: 1482 TYPE: DNA ORGANISM: Cricetulus griseus FEATURE: NAME/KEY: CDS LOCATION: (1) ... (1479)

<4 OOs, SEQUENCE: 3 atg gct gac to a a.a.a. cca citc aat gcc Ctg gat 999 gac cott gtg gct 48 Met Ala Asp Ser Lys Pro Lieu. Asn Ala Luell Asp Gly Asp Pro Wall Ala 1. 1O 15 gtg gag to c tta citc. cgg gat gtg titt 999 att gtt gta gat gag gCC 96 Wall Glu Ser Luell Lell Arg Asp Val Phe Gly Ile Wall Wall Asp Glu Ala US 9,068,212 B2 49 50 - Continued

25 att cgg a.a.a. 999 a CC gcc tog gag aag gtt gaa tgg aag gag 144 Ile Arg Lys Gly Thir Ser Ala Ser Glu Lys Wall Glu Trp Lys Glu 35 4 O 45 cott gala gag citc. aag Cat Ctg gat ttg gag Ctg Cag agc cag ggc 192 Pro Glu Glu Luell His Lell Luell Asp Luell Glu Lell Glin Ser Glin Gly SO 55 6 O gag tot Cala gag Cag att Cta gag cgc tgc cgg gct gtg att CaC tac 24 O Glu Ser Glin Glu Glin Ile Lell Glu Arg Cys Arg Ala Wall Ile His Tyr 65 70 7s 8O

gt C aag act ggit CaC c cc cgg ttic ttic aac Cag citc. ttic to a 999 288 Ser Wall Lys Thir Gly His Pro Arg Phe Phe ASn Glin Lell Phe Ser Gly 85 90 95 tta gac cc c Cat gct Ctg gct 999 cgc at C atc. a Ca gaa agc citc. aac 336 Lell Asp Pro His Ala Lell Ala Gly Arg Ile Ile Thir Glu Ser Luell Asn 1OO 105 11 O a CC agc cag tac a Ca gag att gcc cott gtg titt gtc citc. atg gala 384 Thir Ser Glin Tyr Thir Glu Ile Ala Pro Wall Phe Wall Luell Met Glu 115 12 O 125 gag gag gtg Ctg aag citc. cgt gcc Ctg gtg ggc tgg aac tot 999 432 Glu Glu Wall Luell Lys Lell Arg Ala Luell Wall Gly Trp Asn Ser Gly 13 O 135 14 O gat 999 gt C ttic tgt cott ggit ggc to c at C tog aac atg tat gcc atg Asp Gly Wall Phe Cys Pro Gly Gly Ser Ile Ser Asn Met Ala Met 145 150 155 160 aac Ctg gcc cgc tat Cag cgc tac CC a gac tgc aag Cala aga ggc citc. 528 Asn Luell Ala Arg Tyr Glin Arg Pro Asp Cys Lys Glin Arg Gly Luell 1.65 170 175 cgg gcc Ctg cc.g c cc ttg gct citc. ttic act toa aag gag tgt CaC tac 576 Arg Ala Luell Pro Pro Lell Ala Luell Phe Thir Ser Lys Glu Cys His 18O 185 19 O t cc at C agt aag gga gct gct titt gga citt ggc act gac agt gt C 624 Ser Ile Ser Lys Gly Ala Ala Phe Luell Gly Luell Gly Thir Asp Ser Wall 195 2OO 2O5 cga gtg gt C aag gct gat gag aga 999 a.a.a. atg atc. cott gag gat Ctg 672 Arg Wall Wall Lys Ala Asp Glu Arg Gly Lys Met Ile Pro Glu Asp Luell 21 O 215 22O gag agg cag at C agt Ctg gct gag gca gag ggc tot gtg CC a titt Ctg 72 O Glu Arg Glin Ile Ser Lell Ala Glu Ala Glu Gly Ser Wall Pro Phe Luell 225 23 O 235 24 O gtc agt acc acc tot ggit a CC acc gtg Cta 999 gcc titt gac cc c 768 Wall Ser Thir Thir Ser Gly Thir Thir Wall Luell Gly Ala Phe Asp Pro Luell 245 250 255 gat gca att gct gat gtt tgc cag cgt CaC gga tta tgg tta CaC gtg 816 Asp Ala Ile Ala Asp Wall Cys Glin Arg His Gly Lell Trp Luell His Wall 26 O 265 27 O gat gcc gcc tgg ggit 999 agc gt C Ctg Ctg to c cgg a Ca CaC agg Cat 864 Asp Ala Ala Trp Gly Gly Ser Wall Luell Luell Ser Arg Thir His Arg His 27s 28O 285 citc. Ctg gat 999 atc. Cag agg gct gac tot gtg gcc tgg aac cott CaC 912 Lell Luell Asp Gly Ile Glin Arg Ala Asp Ser Wall Ala Trp Asn Pro His 29 O 295 3 OO aag citt citc. ggt gca 999 Ctg cag tot gct citt citt citc. cgg gac 96.O Lys Luell Luell Gly Ala Gly Lell Glin Ser Ala Lell Lell Luell Arg Asp 3. OS 310 315 32O a CC tog aac Ctg citc. aag cgc Cat 999 to c Cag gcc agc tac Ctg 1008 Thir Ser Asn Luell Lell Arg His Gly Ser Glin Ala Ser Tyr Luell 3.25 330 335 tto cag cag gac a.a.a. tto tat gac gct citt gac act gga gac aag 1056 US 9,068,212 B2 51 52 - Continued

Phe Glin Glin Asp Lys Phe Asp Wall Ala Luell Asp Thir Gly Asp Lys 34 O 345 35. O gtg gtg cag tgt ggc cgc cgt gtg gac tgt Ctg aag ttg tgg citc. atg 104 Wall Wall Glin Cys Gly Arg Arg Wall Asp Cys Luell Lys Lell Trp Luell Met 355 360 365 tgg aag gca cag ggit 999 Cala gga Ctg gag cgg cgc atc. gac cag gcc 152 Trp Lys Ala Glin Gly Gly Glin Gly Luell Glu Arg Arg Ile Asp Glin Ala 37 O 375 38O titt gct citc. acc cgg tac Ctg gtg gag gag ata a.a.a. aag cgg gala gga 2OO Phe Ala Luell Thir Tyr Lell Wall Glu Glu Ile Lys Arg Glu Gly 385 390 395 4 OO titt gag ttg gt C atg gag cott gag titt gt C aat gtg tgc ttic tgg titt 248 Phe Glu Luell Wall Met Glu Pro Glu Phe Wall ASn Wall Cys Phe Trp Phe 4 OS 41O 415 gtg cott cc c agc Ctg cgg 999 aag a.a.a. gag agt C Ca gat tac agc a.a.a. 296 Wall Pro Pro Ser Lell Arg Gly Lys Lys Glu Ser Pro Asp Tyr Ser Lys 42O 425 43 O agg Ctg tot cag gtg gcg cott gta citc. aag gag cgc atg gtg aag aag 344 Arg Luell Ser Glin Wall Ala Pro Wall Luell Lys Glu Arg Met Wall Lys Lys 435 44 O 445 ggc to c atg atg att ggc tac cag cc c Cat 999 a CC cgg gcc aac ttic 392 Gly Ser Met Met Ile Gly Tyr Glin Pro His Gly Thir Arg Ala Asn Phe 450 45.5 460 tto cgg atg gtg gtg gcc aac cc c aca Ctg acc Cag gct gat at a gac 44 O Phe Arg Met Wall Wall Ala Asn Pro Thir Luell Thir Glin Ala Asp Ile Asp 465 470 47s 48O tto citt Ctg ggc gag Ctg gag cgt Ctg ggc cag gac Ctg tga 482 Phe Lieu Lieu Gly Glu Lieu Glu Arg Lieu Gly Gln Asp Lieu 485 490

<210s, SEQ ID NO 4 &211s LENGTH: 493 212. TYPE : PRT &213s ORGANISM: Cricetulus griseus

<4 OOs, SEQUENCE: 4.

Met Ala Asp Ser Lys Pro Lell Asn Ala Luell Asp Gly Asp Pro Wall Ala 1. 5 1O 15

Wall Glu Ser Luell Lell Arg Asp Wall Phe Gly Ile Wall Wall Asp Glu Ala 25

Ile Arg Lys Gly Thir Ser Ala Ser Glu Wall Glu Trp Glu 35 4 O 45

Pro Glu Glu Luell His Lell Luell Asp Luell Glu Lell Glin Ser Glin Gly SO 55 6 O

Glu Ser Glin Glu Glin Ile Lell Glu Arg Arg Ala Wall Ile His Tyr 65 70

Ser Wall Thir Gly His Pro Arg Phe Phe ASn Glin Lell Phe Ser Gly 85 90 95

Lell Asp Pro His Ala Lell Ala Gly Arg Ile Ile Thir Glu Ser Luell Asn 105 11 O

Thir Ser Glin Tyr Thir Glu Ile Ala Pro Wall Phe Wall Luell Met Glu 115 12 O 125

Glu Glu Wall Luell Lell Arg Ala Luell Wall Gly Trp Asn Ser Gly 13 O 135 14 O

Asp Gly Wall Phe Pro Gly Gly Ser Ile Ser Asn Met Ala Met 145 150 155 160

Asn Luell Ala Arg Tyr Glin Arg Pro Asp Glin Arg Gly Luell 1.65 17O 17s US 9,068,212 B2 53 54 - Continued

Arg Ala Luell Pro Pro Leu Ala Lieu. Phe Thir Ser Lys Glu Cys His Tyr 18O 185 19 O

Ser Ile Ser Lys Gly Ala Ala Phe Lieu. Gly Lieu. Gly Thir Asp Ser Wall 195

Arg Wall Wall Lys Ala Asp Glu Arg Gly Lys Met Ile Pro Glu Asp Lieu. 21 O 215

Glu Arg Glin Ile Ser Lieu. Ala Glu Ala Glu Gly Ser Wall Pro Phe Lieu. 225 23 O 235 24 O

Wall Ser Thir Thir Ser Gly. Thir Thr Val Lieu. Gly Ala Phe Asp Pro Leu 245 250 255

Asp Ala Ile Ala Asp Val Cys Glin Arg His Gly Lell Trp Luell His Wall 26 O 265 27 O

Asp Ala Ala Trp Gly Gly Ser Val Lieu. Luell Ser Arg Thir His Arg His 285

Lell Luell Asp Gly Ile Glin Arg Ala Asp Ser Val Ala Trp Asn Pro His 29 O 295 3 OO

Lys Luell Luell Gly Ala Gly Lieu. Glin Cys Ser Ala Lell Lell Luell Arg Asp 3. OS 310 315 32O

Thir Ser Asn Lieu Lleu Lys Arg Cys His Gly Ser Glin Ala Ser Tyr Lieu. 3.25 330 335

Phe Glin Glin Asp Llys Phe Tyr Asp Wall Ala Lieu. Asp Thir Gly Asp Llys 34 O 345 35. O

Wall Wall Glin Cys Gly Arg Arg Val Asp Cys Lieu. Lell Trp Luell Met 355 360 365

Trp Lys Ala Glin Gly Gly Glin Gly Lieu. Glu Arg Arg Ile Asp Glin Ala 37 O 375

Phe Ala Luell Thr Arg Tyr Lieu Val Glu Glu Ile Arg Glu Gly 385 390 395 4 OO

Phe Glu Luell Wal Met Glu Pro Glu Phe Wall Asn Wall Phe Trp Phe 4 OS 41O 415

Wall Pro Pro Ser Lieu. Arg Gly Lys Lys Glu Ser Pro Asp Tyr Ser Lys 425 43 O

Arg Luell Ser Glin Wall Ala Pro Wall Lieu Lys Glu Arg Met Wall 435 44 O 445

Gly Ser Met Met Ile Gly Tyr Glin Pro His Gly Thir Arg Ala Asn. Phe 450 45.5 460

Phe Arg Met Wal Wall Ala ASn Pro Thir Lieu. Thir Glin Ala Asp Ile Asp 465 470 47s 48O

Phe Luell Luell Gly Glu Lieu. Glu Arg Lieu. Gly Glin Asp Lell 485 490

<210s, SEQ ID NO 5 &211s LENGTH: 1491 &212s. TYPE: DNA <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 5 atggcctica gcacaggtga ccggagc.cag gC9gtgaggc atggactgag ggcgalaggtg 6 O

Ctgacgctgg acggcatgaa cggaga.gtgg agtacgcagt gcgtggCCCC 12 O at agtgcagc gag ccttgga gctggagcag gagctgcgc.c agggtgttgaa gaagc ctitt c 18O accgaggt ca tcc.gtgccaa catcggggac gcacaggcta tgggg.ca.gag gcc catcacc 24 O titcc togcgcc aggtottggc cct ctdtgtt aac cctdatc ttctgagcag CCC Caactt C 3OO

US 9,068,212 B2 57 58 - Continued

1.65 17O 17s

His Thir Arg Thir Gly Wall Lell Ile Pro Ile Pro Glin Tyr Pro Luell Tyr 18O 185 19 O

Ser Ala Thir Luell Ala Glu Lell Gly Ala Wall Glin Wall Asp Tyr Luell 195 2OO

Asp Glu Glu Arg Ala Trp Ala Luell Asp Wall Ala Glu Lell His Arg Ala 21 O 215

Lell Gly Glin Ala Arg Asp His Arg Pro Arg Ala Lell Wall Ile 225 23 O 235 24 O

Asn Pro Gly Asn Pro Thir Gly Glin Wall Glin Thir Arg Glu Ile Glu 245 250 255

Ala Wall Ile Arg Phe Ala Phe Glu Glu Arg Luell Phe Lell Luell Ala Asp 26 O 265 27 O

Glu Wall Tyr Glin Asp Asn Wall Tyr Ala Ala Gly Ser Glin Phe His Ser 27s 285

Phe Lys Wall Lell Met Glu Met Gly Pro Pro Tyr Ala Gly Glin Glin 29 O 295 3 OO

Glu Luell Ala Ser Phe His Ser Thir Ser Gly Met Gly Glu Cys 3. OS 310 315

Gly Phe Arg Gly Gly Tyr Wall Glu Wall Wall ASn Met Asp Ala Ala Wall 3.25 330 335

Glin Glin Glin Met Lell Lell Met Ser Wall Arg Lell Pro Pro Wall 34 O 345 35. O

Pro Gly Glin Ala Lell Lell Asp Luell Wall Wall Ser Pro Pro Ala Pro Thir 355 360 365

Asp Pro Ser Phe Ala Glin Phe Glin Ala Glu Lys Glin Ala Wall Luell Ala 37 O 375

Glu Luell Ala Ala Ala Luell Thir Glu Glin Wall Phe Asn Glu Ala 385 390 395 4 OO

Pro Gly Ile Ser Cys Asn Pro Wall Glin Gly Ala Met Ser Phe Pro 4 OS 415

Arg Wall Glin Luell Pro Pro Arg Ala Wall Glu Arg Ala Glin Glu Luell Gly 425 43 O

Lell Ala Pro Asp Met Phe Phe Cys Luell Arg Luell Lell Glu Glu Thir Gly 435 44 O 445

Ile Cys Wall Wall Pro Gly Ser Gly Phe Gly Glin Arg Glu Gly Thir Tyr 450 45.5 460

His Phe Arg Met Thir Ile Lell Pro Pro Luell Glu Lell Arg Luell Luell 465 470

Lell Luell Ser Arg Phe His Ala Lys Phe Thir Lell Glu Tyr Ser 485 490 495

Met Ser Ser Thir Gly Asp Arg Ser Glin Ala Wall Arg His Gly Luell SOO 505

Arg Lys Wall Lell Thir Lell Asp Gly Met ASn Pro Arg Wall Arg Arg 515 525

Wall Ala Wall Arg Gly Pro Ile Wall Glin Arg Ala Luell Glu Luell 535 54 O

Glu Glu Luell Arg Glin Gly Wall Pro Phe Thir Glu Wall Ile 5.45 550 555 560

Arg Asn Ile Gly Asp Ala Glin Ala Met Gly Glin Arg Pro Ile Thir 565 st O sts

Phe Luell Arg Glin Wall Lell Ala Luell Cys Wall ASn Pro Asp Luell Luell Ser 58O 585 59 O US 9,068,212 B2 59 60 - Continued

Ser Pro Asn Phe Pro Asp Asp Ala Lys Arg Ala Glu Arg Ile Luell 595 605

Glin Ala Gly Gly His Ser Luell Gly Ala Ser Wall Ser Ser Gly 610 615

Ile Glin Luell Ile Arg Glu Asp Wall Ala Arg Tyr Ile Glu Arg Arg Asp 625 630 635 64 O

Gly Gly Ile Pro Ala Asp Pro Asn Asn Wall Phe Lell Ser Thir Gly Ala 645 650 655

Ser Asp Ala Ile Wall Thir Wall Luell Lys Luell Luell Wall Ala Gly Glu Gly 660 665 67 O

His Thir Arg Thir Gly Wall Lell Ile Pro Ile Pro Glin Tyr Pro Luell Tyr 675 685

Ser Ala Thir Luell Ala Glu Lell Gly Ala Wall Glin Wall Asp Luell 69 O. 695 7 OO

Asp Glu Glu Arg Ala Trp Ala Luell Asp Wall Ala Glu Lell His Arg Ala 7 Os

Lell Gly Glin Ala Arg Asp His Arg Pro Arg Ala Lell Wall Ile 72 73 O 73

Asn Pro Gly Asn Pro Thir Gly Glin Wall Glin Thir Arg Glu Cys Ile Glu 740 74. 7 O

Ala Wall Ile Arg Phe Ala Phe Glu Glu Arg Luell Phe Lell Luell Ala Asp 7ss 760 765

Glu Wall Glin Asp Asn Wall Ala Ala Gly Ser Glin Phe His Ser 770 775 78O

Phe Wall Lell Met Glu Met Gly Pro Pro Ala Gly Glin Glin 79 O 79.

Glu Luell Ala Ser Phe His Ser Thir Ser Lys Gly Met Gly Glu Cys 805 810 815

Gly Phe Arg Gly Gly Wall Glu Wall Wall ASn Met Asp Ala Ala Wall 825 83 O

Glin Glin Glin Met Lell Lell Met Ser Wall Arg Lell Cys Pro Pro Wall 835 84 O 845

Pro Gly Glin Ala Lell Lell Asp Luell Wall Wall Ser Pro Pro Ala Pro Thir 850 855 860

Asp Pro Ser Phe Ala Glin Phe Glin Ala Glu Lys Glin Ala Wall Luell Ala 865

Glu Luell Ala Ala Lys Ala Luell Thir Glu Glin Wall Phe Asn Glu Ala 885 890 895

Pro Gly Ile Ser Cys Asn Pro Wall Glin Gly Ala Met Ser Phe Pro 9 OO 905 91 O

Arg Wall Glin Luell Pro Pro Arg Ala Wall Glu Arg Ala Glin Glu Luell Gly 915 92 O 925

Lell Ala Pro Asp Met Phe Phe Luell Arg Luell Lell Glu Glu Thir Gly 93 O 935 94 O

Ile Wall Wall Pro Gly Ser Gly Phe Gly Glin Arg Glu Gly Thir Tyr 945 950 955 96.O

His Phe Arg Met Thir Ile Lell Pro Pro Luell Glu Lell Arg Luell Luell 965 97O 97.

Lell Glu Luell Ser Arg Phe His Ala Phe Thir Lell Glu Ser 98O 985 99 O

<210s, SEQ ID NO 7 &211s LENGTH: 1869

US 9,068,212 B2 65 - Continued

595 6OO 605 gca Ct c atgaaa CCC agt cac git C att gtg gag acc atg atg tda 1869 Ala Leu Met Llys Pro Ser His Val Ile Val Glu Thr Met Met 610 615 62O

<210s, SEQ ID NO 8 &211s LENGTH: 622 212. TYPE: PRT <213> ORGANISM: Cricetulus griseus <4 OOs, SEQUENCE: 8 Met Ala Thir Lys Glu Lys Lieu. Glin Cys Lieu Lys Asp Phe His Lys Asp 1. 5 1O 15 Ile Leu Lys Pro Ser Pro Gly Lys Ser Pro Gly Thr Arg Pro Glu Asp 2O 25 3O Glu Ala Glu Gly Llys Pro Pro Glin Arg Glu Lys Trp Ser Ser Lys Ile 35 4 O 45 Asp Phe Val Lieu. Ser Val Ala Gly Gly Phe Val Gly Lieu. Gly Asn Val SO 55 6 O Trp Arg Phe Pro Tyr Lieu. Cys Tyr Lys Asn Gly Gly Gly Ala Phe Lieu. 65 70 7s 8O Ile Pro Tyr Phe Ile Phe Leu Phe Gly Ser Gly Lieu Pro Val Phe Phe 85 90 95 Lieu. Glu Val Ile Ile Gly Glin Tyr Thr Ser Glu Gly Gly Ile Thr Cys 1OO 105 11 O Trp. Glu Lys Ile Cys Pro Leu Phe Ser Gly Ile Gly Tyr Ala Ser Ile 115 120 125 Val Ile Val Ser Lieu. Lieu. Asn Val Tyr Tyr Ile Val Ile Lieu Ala Trip 13 O 135 14 O Ala Thr Tyr Tyr Lieu Phe His Ser Phe Glin Thr Glu Lieu Pro Trp Ala 145 150 155 160 His Cys Asn His Ser Trp Asn Thr Pro His Cys Met Glu Asp Thr Lieu. 1.65 17O 17s Arg Arg Asn. Glu Ser Lieu. Trp Val Ser Lieu. Ser Ala Ser Asn. Phe Thr 18O 185 19 O Ser Pro Val Ile Glu Phe Trp Glu Arg Asin Val Lieu Ser Leu Ser Ser 195 2OO 2O5 Gly Ile Asp Glu Pro Gly Ala Lieu Lys Trp Asp Lieu Ala Lieu. Cys Lieu. 21 O 215 22O Lieu. Lieu Val Trp Lieu Val Cys Phe Phe Cys Ile Trp Llys Gly Val Arg 225 23 O 235 24 O Ser Thr Gly Llys Val Val Tyr Phe Thr Ala Thr Phe Pro Phe Ala Met 245 250 255 Lieu. Lieu Val Lieu. Lieu Val Arg Gly Lieu. Thir Lieu Pro Gly Ala Gly Glu 26 O 265 27 O Gly Ile Llys Phe Tyr Lieu. Tyr Pro Asp Ile Ser Arg Lieu. Glu Asp Pro 27s 28O 285

Glin Val Trp Ile Asp Ala Gly Thr Glin Ile Phe Phe Ser Tyr Ala Ile 29 O 295 3 OO

Cys Lieu. Gly Ala Met Thir Ser Leu Gly Ser Tyr Asn Lys Tyr Lys Tyr 3. OS 310 315 32O

Asn Ser Tyr Arg Asp Cys Met Lieu. Lieu. Gly Cys Lieu. Asn. Ser Gly Thr 3.25 330 335

Ser Phe Val Ser Gly Phe Ala Val Phe Ser Ile Leu Gly Phe Met Ala 34 O 345 35. O US 9,068,212 B2 67 - Continued

Glin Glu Glin Gly Val Asp Ile Ala Asp Wall Ala Glu Ser Gly Pro Gly 355 360 365 Lieu Ala Phe Ile Ala Tyr Pro Lys Ala Val Thr Met Met Pro Leu Pro 37 O 375 38O Thr Phe Trp Ser Ile Leu Phe Phe Ile Met Leu Lleu Lleu Lieu. Gly Lieu. 385 390 395 4 OO Asp Ser Glin Phe Val Glu Val Glu Gly Glin Ile Thr Ser Leu Val Asp 4 OS 41O 415 Lieu. Tyr Pro Ser Phe Lieu. Arg Lys Gly Tyr Arg Arg Glu Val Phe Ile 42O 425 43 O Ala Ile Lieu. Cys Ser Ile Ser Tyr Lieu. Leu Gly Lieu Ser Met Val Thr 435 44 O 445 Glu Gly Gly Met Tyr Val Phe Gln Leu Phe Asp Tyr Tyr Ala Ala Ser 450 45.5 460 Gly Val Cys Lieu Lleu Trp Val Ala Phe Phe Glu. Cys Phe Val Ile Ala 465 470 47s 48O Trp Ile Tyr Gly Gly Asp Asn Lieu. Tyr Asp Gly Ile Glu Asp Met Ile 485 490 495 Gly Tyr Arg Pro Gly Pro Trp Met Lys Tyr Ser Trp Ala Val Ile Thr SOO 505 51O Pro Val Lieu. Cys Ala Gly Cys Phe Ile Phe Ser Lieu Val Lys Tyr Val 515 52O 525 Pro Leu. Thr Tyr Asn Llys Val Tyr Val Tyr Pro Asp Trp Ala Ile Gly 53 O 535 54 O Lieu. Gly Trp Gly Lieu Ala Lieu. Ser Ser Met Val Cys Ile Pro Lieu Val 5.45 550 555 560 Ile Ala Ile Lieu. Lieu. Cys Arg Thr Glu Gly Pro Phe Arg Val Arg Ile 565 st O sts Glin Tyr Lieu. Ile Thr Pro Arg Glu Pro Asn Arg Trp Ala Val Glu Arg 58O 585 59 O Glu Gly Ala Thr Pro Phe His Ser Arg Thr Ser Lieu Val Met Asin Gly 595 6OO 605 Ala Leu Met Llys Pro Ser His Val Ile Val Glu Thr Met Met 610 615 62O

<210s, SEQ ID NO 9 &211s LENGTH: 6 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic 6xHis tag

<4 OOs, SEQUENCE: 9

His His His His His His 1. 5

<210s, SEQ ID NO 10 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic 1OXHis tag

<4 OOs, SEQUENCE: 10

His His His His His His His His His His 1. 5 1O US 9,068,212 B2 69 - Continued

<210s, SEQ ID NO 11 &211s LENGTH: 911 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 11 Met Glu Glu Lieu. Glin Asp Asp Tyr Glu Asp Met Met Glu Glu Asn Lieu. 1. 5 1O 15 Glu Glin Glu Glu Tyr Glu Asp Pro Asp Ile Pro Glu Ser Glin Met Glu 2O 25 3O Glu Pro Ala Ala His Asp Thr Glu Ala Thr Ala Thr Asp Tyr His Thr 35 4 O 45 Thir Ser His Pro Gly Thr His Llys Val Tyr Val Glu Lieu. Glin Glu Lieu. SO 55 6 O Val Met Asp Glu Lys Asn Glin Glu Lieu. Arg Trp Met Glu Ala Ala Arg 65 70 7s 8O Trp Val Glin Lieu. Glu Glu Asn Arg Gly Glu Asn Gly Ala Trp Gly Arg 85 90 95 Pro His Lieu. Ser His Lieu. Thir Phe Trp Ser Lieu. Lieu. Glu Lieu. Arg Arg 1OO 105 11 O Val Phe Thr Lys Gly Thr Val Lieu. Lieu. Asp Leu Gln Glu Thir Ser Leu 115 12 O 125 Ala Gly Val Ala Asn Gln Lieu. Lieu. Asp Arg Phe Ile Phe Glu Asp Glin 13 O 135 14 O Ile Arg Pro Glin Asp Arg Glu Glu Lieu Lieu. Arg Ala Lieu Lieu. Lieu Lys 145 150 155 160 His Ser His Ala Gly Glu Lieu. Glu Ala Lieu. Gly Gly Val Llys Pro Ala 1.65 17O 17s Val Lieu. Thir Arg Ser Gly Asp Pro Ser Glin Pro Leu Lleu Pro Glin His 18O 185 19 O Ser Ser Lieu. Glu Thr Gln Leu Phe Cys Glu Gln Gly Asp Gly Gly Thr 195 2OO 2O5 Glu Gly His Ser Pro Ser Gly Ile Leu Glu Lys Ile Pro Pro Asp Ser 21 O 215 22O Glu Ala Thir Lieu Val Lieu Val Gly Arg Ala Asp Phe Lieu. Glu Gln Pro 225 23 O 235 24 O Val Lieu. Gly Phe Val Arg Lieu. Glin Glu Ala Ala Glu Lieu. Glu Ala Val 245 250 255 Glu Lieu Pro Val Pro Ile Arg Phe Leu Phe Val Lieu. Leu Gly Pro Glu 26 O 265 27 O Ala Pro His Ile Asp Tyr Thr Glin Lieu. Gly Arg Ala Ala Ala Thir Lieu 27s 28O 285 Met Ser Glu Arg Val Phe Arg Ile Asp Ala Tyr Met Ala Glin Ser Arg 29 O 295 3 OO

Gly Glu Lieu. Lieu. His Ser Lieu. Glu Gly Phe Lieu. Asp Cys Ser Lieu Val 3. OS 310 315 32O

Lieu Pro Pro Thir Asp Ala Pro Ser Glu Glin Ala Lieu Lleu Ser Lieu Val 3.25 330 335

Pro Val Glin Arg Glu Lieu. Lieu. Arg Arg Arg Tyr Glin Ser Ser Pro Ala 34 O 345 35. O

Llys Pro Asp Ser Ser Phe Tyr Lys Gly Lieu. Asp Lieu. Asn Gly Gly Pro 355 360 365

Asp Asp Pro Lieu. Glin Glin Thr Gly Glin Lieu. Phe Gly Gly Lieu Val Arg US 9,068,212 B2 71 72 - Continued

37 O 375

Asp Ile Arg Arg Arg Tyr Pro Luell Ser Asp Ile Thir Asp Ala 385 390 395 4 OO

Phe Ser Pro Glin Wall Lell Ala Ala Wall Ile Phe Ile Phe Ala Ala 4 OS 415

Lell Ser Pro Ala Ile Thir Phe Gly Gly Luell Luell Gly Glu Lys Thir Arg 425 43 O

Asn Glin Met Gly Wall Ser Glu Luell Luell Ile Ser Thir Ala Wall Glin Gly 435 44 O 445

Ile Luell Phe Ala Lell Lell Gly Ala Glin Pro Luell Lell Wall Wall Gly Phe 450 45.5 460

Ser Gly Pro Luell Lell Wall Phe Glu Glu Ala Phe Phe Ser Phe Glu 465 470

Thir Asn Gly Luell Glu Ile Wall Gly Arg Wall Trp Ile Gly Phe Trp 485 490 495

Lell Ile Luell Luell Wall Wall Lell Wall Wall Ala Phe Glu Gly Ser Phe Luell SOO 505

Wall Arg Phe Ile Ser Arg Thir Glin Glu Ile Phe Ser Phe Luell Ile 515 525

Ser Luell Ile Phe Ile Glu Thir Phe Ser Lell Ile Ile Phe 53 O 535 54 O

Glin Asp His Pro Lell Glin Thir Asn Tyr Asn Wall Luell Met Wall 5.45 550 555 560

Pro Pro Glin Gly Pro Lell Pro Asn Thir Ala Lell Lell Ser Luell Wall 565 570 575

Lell Met Ala Gly Thir Phe Phe Phe Ala Met Met Lell Arg Lys Phe Lys 585 59 O

Asn Ser Ser Phe Pro Gly Lys Luell Arg Arg Wall Ile Gly Asp Phe 595 605

Gly Wall Pro Ile Ser Ile Lell Ile Met Wall Luell Wall Asp Phe Phe Ile 610 615

Glin Asp Thir Thir Glin Luell Ser Wall Pro Asp Gly Phe Wall 625 630 635 64 O

Ser Asn Ser Ser Ala Arg Gly Trp Wall Ile His Pro Lell Gly Luell Arg 645 650 655

Ser Glu Phe Pro Ile Trp Met Met Phe Ala Ser Ala Lell Pro Ala Luell 660 665 67 O

Lell Wall Phe Ile Lell Ile Phe Luell Glu Ser Glin Ile Thir Thir Luell Ile 675 685

Wall Ser Pro Gly Arg Lys Met Wall Gly Ser Gly Phe His Luell 69 O. 695 7 OO

Asp Luell Luell Luell Wall Gly Met Gly Gly Wall Ala Ala Luell Phe Gly 7 Os

Met Pro Trp Luell Ser Thir Thir Wall Arg Ser Ala Thir His Ala Asn 72 73 O 73

Ala Luell Thir Wall Met Ala Ser Thir Pro Gly Ala Ala Ala Glin 740 74. 7 O

Ile Glin Glu Wall Lys Glin Arg Ile Ser Gly Lell Lell Wall Ala Wall 7ss 760 765

Lell Wall Gly Luell Ser Lell Met Glu Pro Ile Lell Ser Arg Ile Pro 770 775

Lell Ala Wall Luell Phe Ile Phe Luell Tyr Met Gly Wall Thir Ser Luell 78s 79. 8OO US 9,068,212 B2 73 74 - Continued

Ser Gly Ile Glin Lell Phe Asp Arg Ile Lieu. Lieu. Lieu. Phe Lys Pro Pro 805 810 815

Tyr His Pro Asp Wall Pro Tyr Wall Lys Arg Wall Lys Thir Trp Arg 825 83 O

Met Tyr Luell Phe Thir Gly Ile Glin Ile Ile Lell Ala Wall Luell Trp 835 84 O 845

Wall Wall Ser Thir Pro Ala Ser Luell Ala Luell Pro Phe Wall Lieu. Ile 850 855 860

Lell Thir Wall Pro Leu. Arg Arg Wall Lieu. Luell Pro Leu. Ile Phe Arg Asn 865 87O 88O

Val Glu Lieu. Glin Cys Lieu. Asp Ala Asp Asp Ala Ala Thir Phe Asp 885 890 895

Glu Glu Glu Gly Arg Asp Glu Tyr Asp Glu Wall Ala Met Pro Wall 9 OO 905 91 O

The invention claimed is: (b) a DNA encoding a polypeptide which has an amino acid 1. A method of producing an antibody comprising sequence derived from the amino acid sequence as culturing an isolated mammalian cell which is transfected shown in SEQID NO: 2 by substitution, deletion, addi with a heterologous DNA encoding a bicarbonate trans 25 tion and/or insertion of no more than one to ten amino porter and is transfected with a heterologous DNA acid residues; encoding a desired antibody and allowing the cell to (c) a DNA encoding a polypeptide having 96% or more produce said antibody, amino acid sequence homology with the amino acid wherein the bicarbonate transporter exchanges Cloutside sequence as shown in SEQID NO: 2; and of a plasma membrane for HCOs inside a plasma mem 30 (d) a DNA having the nucleotide sequence as shown in brane, and wherein the bicarbonate transporter is anion SEQ ID NO: 1. exchanger 1 (AE1), anion exchanger 2 (AE2), or anion 6. An isolated mammalian cell which is transfected with a exchanger 3 (AE3). heterologous DNA encoding a bicarbonate transporter and is 2. The method of claim 1, wherein the cell is further trans transfected with a heterologous DNA encoding a desired fected with a DNA encoding a cysteine sulfinic acid decar antibody, boxylase or alanine aminotransferase. 35 3. The method of claim 1, wherein the bicarbonate trans wherein the bicarbonate transporter exchanges Cloutside porter is anion exchanger 1 (AE 1). of a plasma membrane for HCO inside a plasma mem 4. The method of claim 1, wherein the cell is a Chinese brane, and wherein the bicarbonate transporter is anion hamster ovary cell. exchanger 1 (AE 1), anion exchanger 2 (AE2), or anion 5. The method of claim 1, wherein the anion exchanger 1 40 exchanger 3 (AE3). (AE1) is encoded by a DNA of any one of the following (a) to 7. The cell of claim 6, wherein the cell is further transfected (d): with a DNA encoding a cysteine sulfinic acid decarboxylase (a) a DNA encoding a polypeptide having the amino acid or alanine aminotransferase. sequence as shown in SEQID NO: 2; k k k k k