Microsoft Word 曹东艳.doc

Size: px

Start display at page:

Download "Microsoft Word 曹东艳.doc"

除萧
5 years ago
Views:

1 4 4 Vol. 4 No Journal of Food Safety and Quality Aug., 2013 GAP 曹东艳, 柳 * 倩, 贺晓云, 梅晓宏 (, ) 摘要 : (paox1), (pgap),, pgap, 关键词 : ; ; GAP ; ; Progress in GAP promoter derived constitutive expression system of Pichia pastoris CAO Dong-Yan, LIU Qian, HE Xiao-Yun, MEI Xiao-Hong * (College of Food Science and Nutritional Engineering, China Agricultural University, Beijing , China) ABSTRACT: The Pichia pastoris expression system, which is one of the eukaryotic expression systems for heterologous gene expression, is widely used at present. Compared with the alcohol oxidase I promoter (paox1) derived inductive expression system, the glyceraldehyde-3-phosphate dehydrogenase promoter(pgap) derived constitutive expression system of Pichia pastoris avoids the use of methanol, needs shorter period, and adopts continuous fermentation mode, thus it becomes a hot point in recent researches. The construction of pgap expression vectors, carbon sources, gene copy number, signal sequence, and high-density cell culture of this expression system were summarized in this paper to provide references for further study and application of this expression system in protein expression fields. KEY WORDS: Pichia pastoris; expression system; GAP promoter; heterologous proteins; constitutive expression 20 80,, ;, [1] 2005, 500 [2] paox1 * 通讯作者 :,, xhmei0719@hotmail.com *Corresponding author: MEI Xiao-Hong, Associate Professor, College of Food Science and Nutritional Engineering, China Agricultural Universary, No.17, Qinghua East Road, Haidian District, Beijing , China. xhmei0719@hotmail.com

2 [3],, [4],,,,, paox1 1997, Waterham [5] pgap, pgap,, pgap pgap, 1 pgap 表达载体的构建 1997, Waterham [5] GAP pgap, pgap phil-a1 paox1 phwo10 pgap, Waterham β- phwo10,, β- Sears [6] pgap, puc19, phwo10 pgap, pib1 pib2, β- pib2, β- [7] B(CPB), pib2 pgap, ppic9k-procpb paox1 pgap9k-procpb, Invitrogen pgapz, (pgapz A, pgapz B, pgapz C) (pgapz αa, pgapz αb and pgapz αc),,, Tang [8] pgapzα, Delroisse [9] pgapz B pgapz αa,, Invitrogen,, pgap ppic9k paox1 pgap9k,, [10] 2 毕赤酵母 pgap 表达系统的碳源,, paox1,, [4], Jungo [11] (57%) (43%), 1.3 Xie [12], (191 mg/l), (141 mg/l), 108 mg/l, (52 mg/l),, pgap [5],,, [5,13] pgap, [14-16], [17,18] paox1,, pgap,,,

3 4, : GAP 毕赤酵母 pgap 表达系统的基因拷贝数 Pichia pastoris, 0.4 g/l [19],,,, [20] pgap, Vassileva [21] Zeocin,, 4 4 Hohenblum [14] pgap,, Daly [22], [23], 1.6,, G418 zeocin, Southern blot real-time PCR [24] [25], mrna,,, 4 毕赤酵母 pgap 表达系统的信号序列,, : paox1 α- Zhou [26] α- ppiczα B NS1 Yu [27] α- ppic9k, 365 mg/l pgap, α-, α- Zhang [13] pgap9 K α-, GS115, 176 mg/l Wang [28] α-, α-, Olezdzka [29] aqualysin I α- Ding [30] α-,,,, 5 毕赤酵母 pgap 表达系统的高密度发酵,,, ph,, (40% 50% w/v), [31], [32] pgap,,,, Zhao [33] (5 L) (800 L), ph

4 1220 4,, 48 h ph, Khasa [34] 2 L pgap -, (4 ) 0.2 h -1, 82 mg/l, (1 ) h -1 4, Goodrick [35], pgap 6 结论 pgap paox1,,,, GAP, paox1,, pgap,,,,,, pgap, 参考文献 [1],. [J]., 2007, 3: Luo JH, You ZL. Study on expression of heterologous gene in Pichia pastoris [J]. Biotechnol Bull, 2007, 3: [2] Macauley-Patrick S, Fazenda ML, McNeil B, et al. Heterologous protein production using the Pichia pastoris expression system [J]. Yeast, 2005, 22(4): [3] Ellis SB, Brust PF, Koutz PJ, et al. Isolation of alcohol oxidase and two other methanol regulatable genes from the yeast Pichia pastoris [J]. Mol Cell Biol, 1985, 5(5): [4] Porro D, Sauer M, Branduardi P, et al. Recombinant protein production in yeast [J]. Mol Biotechnol, 2005, 31(3): [5] 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: [6] Sears IB, O Connor J, Rossanese OW, et al. A versatile set of vectors for constitutive and regulated gene expression in Pichia pastoris [J]. Yeast, 1998, 14: [7]. B Alfimeprase [D]., Zhang PT. Expression of procarboxypeptidase B and a Snake Venom Metalloproteinase Alfimeprase in Pichia pastoris [D]. Xiamen University, [8] Tang SQ, Boehme L, Lam H, et al. Pichia pastoris fermentation for phytase production using crude glycerol from biodiesel production as the sole carbon source [J]. Biochem Eng J, 2009, 43(2): [9] Delroisse JM, Dannau M, Gilsoul JJ, et al. Expression of a synthetic gene encoding a Tribolium castaueum carboxylesterase in Pichia pastoris [J]. Protein Expr Purif, 2005, 42: [10] Zhang AL, Luo JX, Zhang TY, et al. Constitutive expression of human angiostatin in Pichia pastoris using the GAP promoter [J]. Acta Genetica Sin, 2004, 31(6): [11] Jungo C, Schenk J, Pasquier M, et al. A quantitative analysis of the benefits of mixed feeds of sorbitol and methanol for the production of recombinant avidin with Pichia pastoris [J]. J Biotechnol, 2007, 131(1): [12] Xie JL, Zhou QW, Du P, et al. Use of different carbon sources in cultivation of recombinant Pichia pastoris for angiostatin production [J]. Enzyme Microb Technol, 2005, 36: [13] Zhang AL, Zhang TY, Luo JX, et al. Constitutive expression of human angiostatin in Pichia pastoris by high-density cell culture [J]. J Ind Microbiol Biotechnol, 2007, 34: [14] 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: [15] Roman K, Christoph G, Christian P, et al. Constitutive expression of Botrytis aclada laccase in Pichia pastoris [J]. Bioengineered, 2012, 3(4): [16] Jiang XP, Chen P, Yin ML, et al. Constitutive expression, purification and characterization of pectin methylesterase from Asper-

4, : GAP 1221 gillus niger in Pichia pastoris for potential application in the fruit juice industry [J]. J Sci Food Agr, 2013, 93: 375 381. [17] Liu ZW, Yin HX, Yi XP, et al.

Constitutive high-level expression of a codon-optimized β-fructosidase gene from the hyperthermophile Thermotoga maritima in Pichia pastoris [J]. Appl Microbiol Biotechnol, 2013, 97: 1201 1212.

Biotechnology, 1987, 5: 479 485. [20] Scorer CA, Clare JJ, McCombie WR, et al.

[21] Vassileva A, Chugh DA, Swaminathan S, et al. Expression of hepatitis B surface antigen in the methytotropic yeast P. pastoris using the GAP promoter [J]. J Biotechnol, 2001, 88: 21 35.

appa-m [J]., 2006, 22(4): 528 533. Luo HY, Huang HQ, Bai YG, et al. (2006) Improving phytase expression by increasing the gene copy number of appa-m in Pichia pastoris [J].

5 4, : GAP 1221 gillus niger in Pichia pastoris for potential application in the fruit juice industry [J]. J Sci Food Agr, 2013, 93: [17] Liu ZW, Yin HX, Yi XP, et al. Constitutive expression of barley α-amylase in Pichia pastoris by high-density cell culture [J]. Mol Biol Rep, 2012, 39: [18] Menéndez C, Martínez D, Trujillo LE, et al. Constitutive high-level expression of a codon-optimized β-fructosidase gene from the hyperthermophile Thermotoga maritima in Pichia pastoris [J]. Appl Microbiol Biotechnol, 2013, 97: [19] Cregg JM, Tschopp JF, Stillman C, et al. High-level expression and efficient assembly of hepatitis B surface antigen in the methylotrophic yeast, Pichia pastoris [J]. Biotechnology, 1987, 5: [20] Scorer CA, Clare JJ, McCombie WR, et al. Rapid selection using G418 of high copy number transformants of Pichia pastoris for high-level foreign gene expression [J]. Nat Biotechnol, 1994, 12(2): [21] Vassileva A, Chugh DA, Swaminathan S, et al. Expression of hepatitis B surface antigen in the methytotropic yeast P. pastoris using the GAP promoter [J]. J Biotechnol, 2001, 88: [22] 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(2): [23],,,. appa-m [J]., 2006, 22(4): Luo HY, Huang HQ, Bai YG, et al. (2006) Improving phytase expression by increasing the gene copy number of appa-m in Pichia pastoris [J]. Chin J Biotechnol, 2006, 22(4): [24],,,. [J]., 2010, 27(5): Lou RJ, Luo LL, Zhang X. Research progress and prospects on Pichia pastoris [J]. J Biol, 2010, 27(5): [25] Higgins DR, Cregg JM. Introduction to Pichia pastoris [M]. Pichia Protocols: Humana Press, [26] Zhou JM, Tang YX, Fang DY, et al. Secreted expression and purification of dengue 2 virus full-length nonstructural glycoprotein NS1 in Pichia pastoris [J]. Virus Genes, 2006, 33(1): [27] Yu P, Tang YP. Construction of the highly secreted endochitinase Pichia pastoris strain and the optimization of chitin-degrading conditions [J]. Carbohydr Polym, 2012, 89(1): [28] Wang SH, Yang TS, Lin SM, et al. Expression, characterization, and purification of recombinant porcine lactoferrin in Pichia pastoris [J]. Protein Expr Purif, 2002, 25(1): [29] Olezdzka G, Dazbrowski S, Kur J. High-level expression, secretion, and purification of the thermostable aqualysin I from Thermus aquaticus YT-1 in Pichia pastoris [J]. Protein Expr Purif, 2003, 29: [30] Ding JL, Lim EH, Li HF, et al. Expression of recombinant vitellogenin in the yeast Pichia pastoris [J]. Biotechnol Bioeng, 2004, 85: [31] Sreekrishna K, Brankamp RG, Kropp KE, et al. Strategies for optimal synthesis and secretion of heterologous proteins in the methylotrophic yeast Pichia pastoris [J]. Gene, 1997, 190: [32] Li X, He XY, Li ZL, et al. Combined strategies for improving the production of recombinant Rhizopus oryzae lipase in Pichia pastoris [J]. BioResources, 2013, 8(2): [33] Zhao W, Wang J, Deng R, et al. Scale-up fermentation of recombinant Candida rugosa lipase expressed in Pichia pastoris using the GAP promoter [J]. J Ind Microbiol Biotechnol, 2008, 35(3): [34] Khasa YP, Khushoo A, Srivastava L, et al. Kinetic studies of constitutive human granulocyte-macrophage colony stimulating factor (hgm-csf) expression in continuous culture of Pichia pastoris [J]. Biotechnol Lett, 2007, 29(12): [35] Goodrick JC, Xu M, Finnegan R, et al. High-level expression and stabilization of recombinant human chitinase produced in a continuous constitutive Pichia pastoris expression system [J]. Biotechnol Bioeng, 2001, 74: 作者简介 ( 责任编辑 : 赵静 ) 曹东艳, 硕士研究生, 主要研究方向为食品生物技术 caody2012@126.com 梅晓宏, 副教授, 主要研究方向为食品生物技术及转基因食品的安全性 xhmei0719@hotmail.com

Microsoft Word 聂雪梅.doc

Microsoft Word 聂雪梅.doc 4 4 Vol. 4 No. 4 2013 8 Journal of Food Safety and Quality Aug., 2013 聂雪梅 *, 李立, 孙利, 高飞 (, 100123) 摘要 : 2011 12,, 2007~2011,,,, 关键词 : ; ; ; ; Trade and notification situation on food import and export