药学学报, 2014, 49(12): 1644-1649
引用本文:
王庆华, 高丽丽, 梁会超, 巩婷, 杨金玲, 朱平. 影响毕赤酵母高效表达重组蛋白的主要因素及其研究进展[J]. 药学学报, 2014, 49(12): 1644-1649.
WANG Qing-hua, GAO Li-li, LIANG Hui-chao, GONG Ting, YANG Jin-ling, ZHU Ping. Research advances of the influence factors of high level expression of recombinant protein in Pichia pastoris[J]. Acta Pharmaceutica Sinica, 2014, 49(12): 1644-1649.

影响毕赤酵母高效表达重组蛋白的主要因素及其研究进展
王庆华, 高丽丽, 梁会超, 巩婷, 杨金玲, 朱平
中国医学科学院、北京协和医学院药物研究所, 天然药物活性物质与功能国家重点实验室, 卫生部天然药物生物合成重点实验室, 北京 100050
摘要:
毕赤酵母是现代分子生物学研究中最重要的重组蛋白表达系统之一.该系统具有表达水平高、遗传稳定性好、可分泌表达、易于高密度发酵和成本低廉等优点.影响重组蛋白在毕赤酵母中高效表达的主要因素有基因拷贝数、密码子偏爱性、启动子、分子伴侣、信号肽、糖基化修饰和发酵工艺等.本文对影响毕赤酵母高效表达的主要因素及其研究进展进行综述, 为进一步提高重组蛋白在毕赤酵母中的表达水平提供借鉴.
关键词:    毕赤酵母      表达系统      重组蛋白      基因表达      分子伴侣     
Research advances of the influence factors of high level expression of recombinant protein in Pichia pastoris
WANG Qing-hua, GAO Li-li, LIANG Hui-chao, GONG Ting, YANG Jin-ling, ZHU Ping
State Key Laboratory of Bioactive Substance and Function of Natural Medicines and Key Laboratory of Biosynthesis of Natural Products of National Health and Family Planning Commission, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China
Abstract:
Pichia pastoris is one of the most important systems used in the field of molecular biology for the expression of recombinant proteins. The system has advantages of high expression, high stability, high secretion, easy high-density fermentation and low cost. Many factors affect the expression of recombinant protein, such as gene copy number, codon usage preference, type of promoter, molecular chaperones, glycosylation, signal peptide and fermentation process. In this review, research advances of the above aspects are summarized, which lay a foundation for improving the expression of recombinant proteins in P.pastoris.
Key words:    Pichia pastoris    expression system    recombinant protein    gene expression    molecular chaperones    
收稿日期: 2014-06-20
基金项目: 国家自然科学基金资助项目(30873384);北京市自然科学基金资助项目(7082064).
通讯作者: 杨金玲, 朱平
Email: yangjl@imm.ac.cn;zhuping@imm.ac.cn
相关功能
PDF(301KB) Free
打印本文
0
作者相关文章
王庆华  在本刊中的所有文章
高丽丽  在本刊中的所有文章
梁会超  在本刊中的所有文章
巩婷  在本刊中的所有文章
杨金玲  在本刊中的所有文章
朱平  在本刊中的所有文章

参考文献:
[1] Gellissen G, Kunze G, Gaillardin C, et al. New yeast expression platforms based on methylotrophic Hansenula polymorpha and Pichia pastoris and on dimorphic Arxula adeninivorans and Yarrowia lipolytica-A comparison [J]. FEMS Yeast Res, 2005, 5: 1079-1096.
[2] Shukla AK, Haase W, Reinhart C, et al. Heterologous expression and comparative characterization of the human neuromedin U subtype II receptor using the methylotrophic yeast Pichia pastoris and mammalian cells [J]. Int J Biochem Cell Biol, 2007, 39: 931-942.
[3] Du CH, Xiao AF. Secretory expression and purification of the recombinant duck interleukin-2 in Pichia pastoris [J]. J Microbiol Biotechnol, 2011, 21: 1264-1269.
[4] Damasceno LM, Pla I, Chang HJ, et al. An optimized fermentation process for high-level production of a single-chain Fv antibody fragment in Pichia pastoris [J]. Protein Expr Purif, 2004, 37: 18-26.
[5] Shi L, Wang D, Chan W, et al. Efficient expression and purification of human interferon alpha2b in the methylotrophic yeast Pichia pastoris [J]. Protein Expr Purif, 2007, 54: 220- 226.
[6] Walsh G. Biopharmaceutical benchmarks 2010 [J]. Nat Biotechnol, 2010, 28: 917-921.
[7] Clare JJ, Rayment FB, Ballantyne SP, et al. High-level expression of tetanus toxin fragment C in Pichia pastoris strains containing multiple tandem integrations of the gene [J]. Nat Biotechnol, 1991, 9: 455-460.
[8] Vassileva A, Chugh DA, Swaminathan S, et al. Expression of hepatitis B surface antigen in the methylotrophic yeast Pichia pastoris using the GAP promoter [J]. J Biotechnol, 2001, 88: 21-35.
[9] Hohenblum H, Gasser B, Maurer M, et al. Effects of gene dosage, promoters, and substrates on unfolded protein stress of recombinant Pichia pastoris [J]. Biotechnol Bioeng, 2004, 85: 367-375.
[10] Zhu T, Guo M, Zhuang Y, et al. Understanding the effect of foreign gene dosage on the physiology of Pichia pastoris by transcriptional analysis of key genes [J]. Appl Microbiol Biotechnol, 2011, 89: 1127-1135.
[11] Lee TS, Krupa RA, Zhang F, et al. BglBrick vectors and datasheets: a synthetic biology platform for gene expression [J]. J Biol Eng, 2011, 5: 1-14.
[12] Marx H, Mecklenbräuker A, Gasser B, et al. Directed gene copy number amplification in Pichia pastoris by vector integration into the ribosomal DNA locus [J]. FEMS Yeast Res, 2009, 9: 1260-1270.
[13] Han XQ, Liu XT, Zhang YG, et al. Codon optimization and expression in Pichia pastoris of E2 gene of classical swine fever virus [J]. Acta Microbiol Sin (微生物学报), 2003, 43: 560-568.
[14] Woo JH, Liu YY, Mathias A, et al. Gene optimization is necessary to express a bivalent anti-human anti-T cell immunotoxin in Pichia pastoris [J]. Protein Expr Purif, 2002, 25: 270-282.
[15] Hu S, Li L, Qiao J, et al. Codon optimization, expression and characterization of an internalizing anti-ErbB2 single chain antibody in Pichia pastoris [J]. Protein Expr Purif, 2006, 47: 249-257.
[16] Huang H, Yang P, Luo H, et al. High-level expression of a truncated 1, 3-1, 4-β-D-glucanase from Fibrobacter succinogenes in Pichia pastoris by optimization of codons and fermentation [J]. Appl Microbiol Biotechnol, 2008, 78: 95-103.
[17] Werten MWT, Van den Bosch TJ, Wind RD, et al. High-yield secretion of recombinant gelatins by Pichia pastoris [J]. Yeast, 1999, 15: 1087-1096.
[18] Lu Y, Jiang L. The strategies for efficient expression of recombinant protein in Pichia pastoris [J]. Prog Microbiol Immunol (微生物学免疫学进展), 2013, 41: 73-79.
[19] Ruth C, Zuellig T, Mellitzer A, et al. Variable production windows for porcine trypsinogen employing synthetic inducible promoter variants in Pichia pastoris [J]. Syst Synth Biol, 2010, 4: 181-191.
[20] Gellissen G. Heterologous protein production in methylotro­phic yeasts [J]. Appl Microbiol Biotechnol, 2000, 54: 741- 750.
[21] Koutz P, Davis GR, Stillman C, et al. Structural comparison of the Pichia pastoris alcohol oxidase genes [J]. Yeast, 1989, 5: 167-177.
[22] Kuberl A, Schneider J, Thallinger GG, et al. High quality genome sequence of Pichia pastoris CBS743 [J]. J Biotechnol, 2011, 154: 312-320.
[23] Shen S, Sulter G, Jeffries TW, et al. A strong nitrogen source-regulated promoter for controlled expression of foreign genes in the yeast Pichia pastoris [J]. Gene, 1998, 216: 93-102.
[24] Waterham HR, Digan ME, Koutz PJ, et al. Isolation of the Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase gene and regulation and use of its promoter [J]. Gene, 1997, 186: 37-44.
[25] Stadlmayr G, Mecklenbräuker A, Rothmüller M, et al. Identification and characterization of novel Pichia pastoris promoters for heterologous protein production [J]. J Biotechnol, 2010, 150: 519-529.
[26] Damasceno LM, Huang CJ, Batt CA. Protein secretion in Pichia pastoris and advances in protein production [J]. Appl Microbiol Biotechnol, 2012, 93: 31-39.
[27] Shen Q, Wu M, Wang HB, et al. The effect of gene copy number and co-expression of chaperone on production of albumin fusion proteins in Pichia pastoris [J]. Appl Microbiol Biotechnol, 2012, 96: 763-772.
[28] Sha C, Yu XW, Lin NX, et al. Enhancement of lipase r27RCL production in Pichia pastoris by regulating gene dosage and co-expression with chaperone protein disulfide isomerase [J]. Enzyme Microb Technol, 2013, 53: 438-443.
[29] Damasceno LM, Anderson KA, Ritter G, et al. Cooverex­pression of chaperones for enhanced secretion of a single-chain antibody fragment in Pichia pastoris [J]. Appl Microbiol Biotechnol, 2007, 74: 381-389.
[30] Guerfal M, Ryckaert S, Jacobs PP, et al. Research The HAC1 gene from Pichia pastoris: characterization and effect of its overexpression on the production of secreted, surface displayed and membrane proteins [J]. Microb Cell Fact, 2010, 9: 49-60.
[31] Qin X, Liu C, Zheng L. Efficiency of signal peptide sequence in yeast secretory expression system [J]. Biotechnol Bull (生物技术), 2010, 20: 95-97.
[32] Kang HA, Nam SW, Kwon KS, et al. High-level secretion of human-antitrypsin from Saccharomyces cerevisiae using inulinase signal sequence [J]. J Biotechnol, 1996, 48: 15-24.
[33] Murasugi A, Tohma-Aiba Y. Comparison of three signals for secretory expression of recombinant human midkine in Pichia pastoris [J]. Biosci Biotechnol Biochem, 2001, 65: 2291.
[34] Ghosalkar A, Sahai V, Srivastava A. Secretory expression of interferon-alpha 2b in recombinant Pichia pastoris using three different secretion signals [J]. Protein Expr Purif, 2008, 60: 103-109.
[35] Xiong AS, Peng RH, Li X, et al. Influence of signal peptide sequences on the expression of heterogeneous proteins in Pichia pastoris [J]. Acta Biochim Biophys Sin (生物化学与生物物理学报), 2003, 35: 154-160.
[36] Daly R, Hearn MT. Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and production [J]. J Mol Recognit, 2005, 18: 119-138.
[37] Vervecken W, Kaigorodov V, Callewaert N, et al. In vivo synthesis of mammalian-like, hybrid-type N-glycans in Pichia pastoris [J]. Appl Environ Microb, 2004, 70: 2639-2646.
[38] Li H, Sethuraman N, Stadheim TA, et al. Optimization of humanized IgGs in glycoengineered Pichia pastoris [J]. Nat Biotechnol, 2006, 24: 210-215.
[39] Liu Y, Xie W, Yu H. Enhanced activity of Rhizomucor miehei lipase by deglycosylation of its propeptide in Pichia pastoris [J]. Curr Microbiol, 2014, 68: 186-191.
[40] Han M, Wang X, Yan G, et al. Modification of recombinant elastase expressed in Pichia pastoris by introduction of N-glycosylation sites [J]. J Biotechnol, 2014, 171: 3-7.
[41] Vellanki RN, Potumarthi R, Mangamoori LN. Constitutive expression and optimization of nutrients for streptokinase production by Pichia pastoris using statistical methods [J]. Appl Biochem Biotechnol, 2009, 158: 25-40.
[42] Niu H, Jost L, Pirlot N, et al. A quantitative study of methanol/sorbitol co-feeding process of a Pichia pastoris Mut+/pAOX1-lacZ strain [J]. Microb Cell Fact, 2013, 12: 33.
[43] Hu XQ, Chu J, Zhang Z, et al. Effects of different glycerol feeding strategies on S-adenosyl-l-methionine biosynthesis by PGAP-driven Pichia pastoris overexpressing methionine adenosyltransferase [J]. J Biotechnol, 2008, 137: 44-49.
[44] Wu D, Chu J, Wang Y, et al. Effect of methanol concentration expression of recombinant human interferon alfacon-1 separation rate of Pichia pastoris [J]. Chin J Biotechnol (生物工程学报), 2011, 12: 1789-1796.
[45] Lee JH, Lee SG, Do H, et al. Optimization of the pilot-scale production of an ice-binding protein by fed-batch culture of Pichia pastoris [J]. Appl Microbiol Biotechnol, 2013, 97: 3383-3393.
[46] Kong N, Mu X, Han H, et al. Pilot-scale fermentation, purification, and characterization of recombinant human Oncostatin M in Pichia pastoris [J]. Protein Expr Purif, 2009, 63: 134-139.
[47] Zhang Y, Teng D, Mao R, et al. Expression of a plectasin-derived peptide NZ2114 in Pichia pastoris and its pharmaco­dynamics, postantibiotic and synergy against Staphylococcus aureus [J]. Appl Microbiol Biotechnol, 2014, 98: 681- 694.
相关文献:
1.杨金玲, 高丽丽, 朱 平, 侯 琦, 王 芬, 于文博, 聂 涛.蝎毒镇痛活性肽基因BmK AngM1的密码子优化及其真核表达分析[J]. 药学学报, 2012,47(10): 1389-1393
2.杨金玲 何惠霞 朱慧新 程克棣 朱 平.蝎毒镇痛活性肽在毕赤酵母中表达条件的优化[J]. 药学学报, 2009,44(1): 91-94