De¢Cient Strains of the Methylotrophic Yeast Ogataea Minuta
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RESEARCH ARTICLE Antibody expression in protease-de¢cient strains ofthe methylotrophic yeast Ogataea minuta Kousuke Kuroda1,2, Yoshinori Kitagawa1, Kazuo Kobayashi1, Haruhiko Tsumura1, Toshihiro Komeda3, Eiji Mori4, Kazuhiro Motoki4, Shiro Kataoka4, Yasunori Chiba5 & Yoshifumi Jigami2,5 1Kirin Brewery Co. Ltd, CMC R&D Laboratories, Gunma, Japan; 2Graduate School of Life and Environmental Science, University of Tsukuba, Ibaraki, Japan; 3Kirin Brewery Co. Ltd, Central Laboratories for Frontier Technology, Kanagawa, Japan; 4Kirin Brewery Co. Ltd, Pharmaceutical Research Laboratories, Gunma, Japan; and 5National Institute of Advanced Industrial Science and Technology (AIST), Ibaraki, Japan Downloaded from https://academic.oup.com/femsyr/article/7/8/1307/549124 by guest on 27 September 2021 Correspondence: Yoshifumi Jigami, National Abstract Institute of Advanced Industrial Science and Technology (AIST), Ibaraki 305-8566, Japan. When human antibody genes were expressed in the methylotrophic yeast Ogataea Tel.: 181 29 861 6160; fax: 181 29 861 minuta, the secreted antibody became partially degraded. To suppress the 6161; e-mail: [email protected] degradation, a vacuolar protease-deficient strain was constructed and its antibody production was evaluated. Although antibody productivity was improved in the Received 28 February 2007; revised 28 May vacuolar protease-deficient strain, the secreted antibody still became partially 2007; accepted 16 June 2007. degraded. Peptide sequencing revealed that the cleavage occurred in the CH1 First published online 23 August 2007. region of the heavy chain, implying that the cleavage was caused by an aspartic protease, Yps1p. To inhibit this cleavage, Yps1p-deficient strains were constructed DOI:10.1111/j.1567-1364.2007.00291.x and their antibody production was evaluated. As a result, the partial degradation of the antibody was suppressed in the O. minuta multiple-protease-deficient strains. Editor: Hyun Kang Keywords Ogataea minuta ; antibody; protease; YPS1 ; PEP4 ; PRB1 . body-dependent cellular cytotoxicity (ADCC) activity are Introduction expected to be produced by yeast that does not have Owing to advances in the technology available to obtain fucosyltransferase activity, because the removal of fucose chimeric antibodies and completely human antibodies from N-linked sugar chains of the antibody leads to high (Ishida et al., 1998), monoclonal antibodies for pharmaceu- ADCC activity (Shinkawa et al., 2003; Kanda et al., 2006). In ticals have gained a great deal of attention. In the mass fact, some researchers have already confirmed that antibo- production of therapeutic antibodies, mammalian cells have dies derived from engineered yeast have a higher affinity for been adopted as conventional hosts, because antibodies the Fcg receptor than those from the native yeast (Li et al., derived from mammalian cells have some modifications, 2006). such as glycosylation, that are similar to those in antibodies Regarding protein expression in yeast, one study achieved from humans. However, the production of these antibodies a secretion yield of 1.2 g LÀ1 in the production of ‘scFv’, using mammalian cells poses potential problems, due to the which is composed of the variable regions of a light chain, cultivation cost. To overcome these problems, alternative VL, and a heavy chain, VH, combined through a flexible production systems using other hosts, such as transgenic linker peptide (Freyre et al., 2000). In addition, ‘Fab’ is also plants, animals, and microorganisms, have been under successfully expressed in yeast that contains both the heavy investigation (Bardor et al., 2003; Ward et al., 2004). chain domains, VH1-CH1, and the entire light chain, VL- In these trials, methylotrophic yeasts have been recog- CL, connected via disulfide bonds (Joosten et al., 2003; nized as an attractive host for the production of recombi- Gasser et al., 2006). Furthermore, engineered yeast to which nant glycoproteins, including antibodies, because they have chaperones, such as disulfide isomerase, were introduced the potential for modifications, such as sugar chain addi- showed increased secretion titers of the antibody fragments tions after protein synthesis, and also have a high ability to (Shusta et al., 1998; Gasser et al., 2006). However, antibody secrete protein. Moreover, antibodies that have higher anti- fragments are not always secreted in large quantities, and FEMS Yeast Res 7 (2007) 1307–1316 c 2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved 1308 K. Kuroda et al. Table 1. Ogataea minuta strains used in this study Strain Genotype Harboring plasmids Source TK3-A Doch1Dura3 Kuroda et al. (2006) TK5-3 Doch1Dura3Dade1 This study TK5-3-L Doch1Dura3Dade1 sL/pOMGPA1DSp This study TK5-3-LH Doch1Dura3Dade1 sL/pOMGPA1DSp, sH/pOMGPU1DSp This study TK9 Doch1Dpep4Dprb1Dura3Dade1 This study TK9-L Doch1Dpep4Dprb1Dura3Dade1 sL/pOMGPA1DSp This study TK9-LH Doch1Dpep4Dprb1Dura3Dade1 sL/pOMGPA1DSp, sH/pOMGPU1DSp This study YK4 Doch1Dpep4Dprb1Dyps1Dura3Dade1 This study YK4-L Doch1Dpep4Dprb1Dyps1Dura3Dade1 sL/pOMGPA1DSp This study YK4-LH Doch1Dpep4Dprb1Dyps1Dura3Dade1 sL/pOMGPA1DSp, sH/pOMGPU1DSp This study Downloaded from https://academic.oup.com/femsyr/article/7/8/1307/549124 by guest on 27 September 2021 YK6 Doch1Dyps1Dura3Dade1 This study YK6-L Doch1Dyps1Dura3Dade1 sL/pOMGPA1DSp This study YK6-LH Doch1Dyps1Dura3Dade1 sL/pOMGPA1DSp, sH/pOMGPU1DSp This study their secretion titers vary depending on the amino acid apparatus. The Dyps1 strain is also thought to be useful for sequences. protein production in yeast. There are several other reports on full-length antibody Previously, we constructed the yeast Ogataea minuta production in yeast (Wood et al., 1985; Horwitz et al., 1988; host–vector system and achieved success in breeding the Li et al., 2006), although the productivity is low or not yeast producing a Man5GlcNAc2 high-mannose-type sugar mentioned. Moreover, no studies have succeeded in improv- chain as a prototype of a humanized sugar chain by ing full-length antibody productivity in yeast. To develop a disruption of the OmOCH1 gene and the introduction of substitute for mammalian cells for conventional antibody the a-1,2-mannosidase gene (Kuroda et al., 2006). In the production, it is necessary to manipulate yeast genetically present report, we show the usefulness of protease-deficient for the effective production of full-length antibodies. strains in the production of antibodies in the yeast In heterologous protein production in yeast, the target O. minuta. products are often degraded by proteases. Some intracellular proteases degrade not only intracellular proteins but also extracellular proteins during the cultivation or the early Materials and methods stage of purification, due to cell lysis. The contamination Yeast strains and media with proteolytic products complicates the purification pro- cess and reduces the yield and specific activities of the The strains used in this study are listed in Table 1. The yeasts product. Therefore, one effective approach is to construct were cultured at 27 1C in the following media: YPG medium both intracellular and extracellular protease-deficient strains (1% Bacto yeast extract, 2% Bacto peptone, 2% glycerol); SG for protein production. In yeast, such as Saccharomyces medium [0.67% yeast nitrogen base without amino acids cerevisiae, the main intracellular proteolytic activities are (YNB), 1% glycerol]; BYPG medium (2% Bacto peptone, attributed to vacuolar proteases, proteinases A and B, 1% Bacto yeast extract, 1.34% YNB, 0.1 M phosphate buffer, encoded by the PEP4 and PRB1 genes, respectively, which pH 6.0, 1% glycerol); and 2 Â BYPG medium (4% Bacto are known to activate themselves and other proteases, such peptone, 2% Bacto yeast extract, 1.34% YNB, 0.1 M phos- as carboxypeptidase Y (Van Den Hazel et al., 1996). As the phate buffer, pH 6.0, 4% glycerol). Transformants were Dpep4Dprb1 strain has lower vacuolar protease activity than selected on the SG medium plate as auxotrophic markers. the wild-type strain, a higher yield of target proteins was obtained (Gleeson et al., 1998; Komeda et al., 2002). DNA methods On the other hand, some reports have revealed that the Dyps1 strain is effective in the production of secretory Escherichia coli DH5a cells were used for the subcloning of proteins in S. cerevisiae (Copley et al., 1998; Kang et al., the plasmids. The plasmids were prepared with a QIAprep 1998; Kerry-Williams et al., 1998; Bourbonnais et al., 2000). Spin Miniprep Kit (Qiagen) from E. coli DH5a cells. DNA The Yps1p (yapsin 1) encoded by the YPS1 gene is an fragments from agarose gel were recovered with the QIA- aspartic protease that localizes at the plasma membrane quick Gel Extraction Kit (Qiagen). The DNA fragments and has overlapping substrate specificity with intracellular amplified by PCR were subjected to DNA sequence analysis endoprotease Kex2p, which is localized at the late Golgi using a DNA sequencer (Model 3700, ABI). The O. minuta c 2007 Federation of European Microbiological Societies FEMS Yeast Res 7 (2007) 1307–1316 Published by Blackwell Publishing Ltd. All rights reserved Antibody production in yeast 1309 Table 2. DNA primers used to make DNA probes for molecular cloning and to select the knockout strains of ADE1 and protease genes DNA primer Nucleotide sequence Amino acid sequence ADE1 gene. Referred genes: S. cerevisiae (GenBank accession number M61209), Candida maltosa (M58322) PAD5 50-TTYGTNGCNACNGAYMGNATHWSNGCNTAYGAYGTNATHATG-30