hIFNβ-HSA在毕赤酵母中的诱导表达及其降解蛋白酶的鉴定
|
摘要
毕赤酵母是近十年来发展起来的一种真核表达体系,具有表达量高、培养成本低、产物可以分泌到胞外、糖基化程度适中等优点。目前,国内外已有500多种外源蛋白在毕赤酵母中得到表达。但是外源蛋白的降解是毕赤酵母表达体系应用过程中的共性问题,严重影响外源蛋白的积累。
本研究室成功构建了IFNβ与人血清白蛋白融合蛋白(Human IFNβ-HSA fusion protein, hIFNβ-HSA)基因并在毕赤酵母(Pichia pastoris KM71)中表达,经检测融合蛋白分子量约为90 kDa,具有HSA的抗原性,用细胞病变抑制法测定摇瓶发酵液上清中融合蛋白的干扰素活性约为640 IU/mL,并通过对5 L发酵罐发酵过程参数及其过程控制对基因工程菌毕赤酵母表达hIFNβ-HSA影响的研究确定一系列的发酵参数。本论文是在此工作基础上,选取的主要结果如下:
1.通过单因子实验,确定了最佳的与蛋白酶活性相关的营养和环境条件:pH在5.0-6.0,蛋白胨的最佳添加量为6%。Glu、Ala和Arg三种氨基酸的添加会促进融合蛋白的表达。其最佳作用浓度为0.1%。最后确定了EDTA为促进融合蛋白表达的最适蛋白酶抑制剂,其最佳作用浓度为10 mmol/L。根据Box-Benhnken的中心组合试验设计原理,采用三因素三水平的响应面分析方法,考察了pH、Glu浓度和EDTA浓度对融合蛋白表达的影响,结果表明当pH 5.5、添加Glu浓度为0.18%、EDTA浓度为12 mmol/L时,融合蛋白的表达量最高,为24.87 mg/L,相对于对照组提高了61%。
2.通过SDS-PAGE确定融合蛋白hIFNβ-HSA在诱导表达过程中确实发生了降解,降解的主要条带出现在65 kDa;通过Western-Blotting杂交实验,证实了65 kDa的蛋白条带与90 kDa的目的条带在杂交实验中具有相同的蛋白免疫原性,从而确定这条蛋白带为90 kDa目的条带的降解带。通过HPLC证实发酵液中蛋白酶对融合蛋白的降解主要发生在HSA区域。以明胶为底物的SDS-PAGE活性电泳结果表明,诱导表达阶段的发酵上清液和细胞内存在几种蛋白酶。以HSA为底物的SDS-PAGE活性电泳,确定了在发酵液中确实存在一种蛋白酶对融合蛋白进行水解作用。在此基础上,对与标准蛋白分子量Mark进行比较,确定该蛋白酶为蛋白酶B。
The yeast Pichia pastoris is a useful eucaryon expressing system which has developed in 10 years. This system has several good quality , for example, high amouts expression, clutivating low, product can secrete outside, proper glycosylation. At present, already have had more than 500 kind external protein expressed in Pichia pastoris. Proteolytic degradation has been a perpetual problem when yeasts are employed to express recombinant proteins. Alos this problem influent the accumulation of protein.
On the basis of previously work, laboratory constructed the gene of Human IFNβ-HSA fusion protein, hIFNβ-HSA in Pichia pastoris KM71 by technique of gene engineering, which express the 90 kDa protein. And tthrough he effects of fermentative process parameter on cell growth and hIFNβ-HSA expression in 5 L fermenter were studied. On this basis , this research is about the problem of the Proteolytic degradation during the express process, the main results as follows:
1. By the experiment of mono-factor, the main results as follows: under the pH5.0～pH6.0 the exprrssion of the protein is the highest, and 6% peptone was added to culture medium is the best, at the same time, Additions of Glu, Ala, Arg into the medium, 0.1% concentration has the optimization expression. According of the principle of Box-Benhnken, with the method of response surface of the three factors and three levels, the best addition program is pH5.5、Glu 0.18%、EDTA 12 mmol/L in the medium, obtained the amount of protein IFNβ-HSA is 24.87 mg/L, comparing with the blank referenc group, increasing 61%
2.. The analysis of SDS-PAGE and Western-blot showed that 90kDa fusion protein and 65kDa degradation band appeared in the fermentation. By the gelatin-SDS-PAGE, three prteases inside and five proteases outside cell, and which degrdated the pritein. With the method of HPLC indicated that the degradation appeared in the HSA region of recombinant human IFNβ-HSA fusion protein. Applied by SDS-PAGE activity electrophoresis the protease which caused the degradation of fusion protein IFNβ-HSA was proteinase B
本研究室成功构建了IFNβ与人血清白蛋白融合蛋白(Human IFNβ-HSA fusion protein, hIFNβ-HSA)基因并在毕赤酵母(Pichia pastoris KM71)中表达,经检测融合蛋白分子量约为90 kDa,具有HSA的抗原性,用细胞病变抑制法测定摇瓶发酵液上清中融合蛋白的干扰素活性约为640 IU/mL,并通过对5 L发酵罐发酵过程参数及其过程控制对基因工程菌毕赤酵母表达hIFNβ-HSA影响的研究确定一系列的发酵参数。本论文是在此工作基础上,选取的主要结果如下:
1.通过单因子实验,确定了最佳的与蛋白酶活性相关的营养和环境条件:pH在5.0-6.0,蛋白胨的最佳添加量为6%。Glu、Ala和Arg三种氨基酸的添加会促进融合蛋白的表达。其最佳作用浓度为0.1%。最后确定了EDTA为促进融合蛋白表达的最适蛋白酶抑制剂,其最佳作用浓度为10 mmol/L。根据Box-Benhnken的中心组合试验设计原理,采用三因素三水平的响应面分析方法,考察了pH、Glu浓度和EDTA浓度对融合蛋白表达的影响,结果表明当pH 5.5、添加Glu浓度为0.18%、EDTA浓度为12 mmol/L时,融合蛋白的表达量最高,为24.87 mg/L,相对于对照组提高了61%。
2.通过SDS-PAGE确定融合蛋白hIFNβ-HSA在诱导表达过程中确实发生了降解,降解的主要条带出现在65 kDa;通过Western-Blotting杂交实验,证实了65 kDa的蛋白条带与90 kDa的目的条带在杂交实验中具有相同的蛋白免疫原性,从而确定这条蛋白带为90 kDa目的条带的降解带。通过HPLC证实发酵液中蛋白酶对融合蛋白的降解主要发生在HSA区域。以明胶为底物的SDS-PAGE活性电泳结果表明,诱导表达阶段的发酵上清液和细胞内存在几种蛋白酶。以HSA为底物的SDS-PAGE活性电泳,确定了在发酵液中确实存在一种蛋白酶对融合蛋白进行水解作用。在此基础上,对与标准蛋白分子量Mark进行比较,确定该蛋白酶为蛋白酶B。
The yeast Pichia pastoris is a useful eucaryon expressing system which has developed in 10 years. This system has several good quality , for example, high amouts expression, clutivating low, product can secrete outside, proper glycosylation. At present, already have had more than 500 kind external protein expressed in Pichia pastoris. Proteolytic degradation has been a perpetual problem when yeasts are employed to express recombinant proteins. Alos this problem influent the accumulation of protein.
On the basis of previously work, laboratory constructed the gene of Human IFNβ-HSA fusion protein, hIFNβ-HSA in Pichia pastoris KM71 by technique of gene engineering, which express the 90 kDa protein. And tthrough he effects of fermentative process parameter on cell growth and hIFNβ-HSA expression in 5 L fermenter were studied. On this basis , this research is about the problem of the Proteolytic degradation during the express process, the main results as follows:
1. By the experiment of mono-factor, the main results as follows: under the pH5.0～pH6.0 the exprrssion of the protein is the highest, and 6% peptone was added to culture medium is the best, at the same time, Additions of Glu, Ala, Arg into the medium, 0.1% concentration has the optimization expression. According of the principle of Box-Benhnken, with the method of response surface of the three factors and three levels, the best addition program is pH5.5、Glu 0.18%、EDTA 12 mmol/L in the medium, obtained the amount of protein IFNβ-HSA is 24.87 mg/L, comparing with the blank referenc group, increasing 61%
2.. The analysis of SDS-PAGE and Western-blot showed that 90kDa fusion protein and 65kDa degradation band appeared in the fermentation. By the gelatin-SDS-PAGE, three prteases inside and five proteases outside cell, and which degrdated the pritein. With the method of HPLC indicated that the degradation appeared in the HSA region of recombinant human IFNβ-HSA fusion protein. Applied by SDS-PAGE activity electrophoresis the protease which caused the degradation of fusion protein IFNβ-HSA was proteinase B
引文
1.罗竞红,游自立.巴斯德毕赤酵母表达系统在外源基因表达中的研究进展[J].生物技术通报,2007,(3):75-79
2.Daly Rachel,Hearn M T W.Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and production.Journal of Molecular Recognition,2005,18:119-138
3.David R.Higgins,JamesM Cremes.Pichia Protocols.New Jersey:Human Press Inc,1998
4.Macauley-Patrick S,FazendaML,McNeil B,et al.Heterologous protein production using the Pichia pastoris expression system.Yeast,2005,22:249-270
5.Cereghino JL,Cregg JM.Heterlogous protein expression in the methylotrophic yeast Pichia pastoris.FEMS Microbiology Reviews,2000,24:45-66
6.Daly R,Hearn MT.Expression of heterologous proteins in Pichia pastoris a useful experimental tool in protein engineering and production.Journal of molecular recognition,2005,18(2):119-138
7.Kout P,Davis,GR,et al.Structural comparison of the Pichia pastoris alcohol oxidase genes.Yeast,1999,5:167-177
8.Thill,G.P.,Davis,G.R., et al.Positive and negative ejects of multicopy integrated expression vectors on protein expression in Pichia pastoris.Proceedings of the Sixth International Symposium on the Genetics of Microorganisms. Socie¨te¨Franc°aise de Microbiologie,1990,2:477-490
9.Brierley,R.A.Secretion of recombinant human insulin-like growth factor I (IGF-1).Methods Mol.Biol.1998,103:149-177
10 . Cregg,J.M. and Madden,K.R.Development of yeast transformation systems and construction of methanol-utilization-defective mutants of Pichia pastoris by gene disruption.Biological Research on Industrial Yeasts(Stewart, G.G., Russell, I., Klein, R.D.and Hiebsch,R.R.,Eds.).1987,2:1-18
11.Chiruvolu,V.,Cregg,J.M. and Meagher,M.M.Recombinant protein production in an alcohol oxidase-defective strain of Pichia pastoris in fed-batch fermentations.Enzyme Microb.Technol.1997,21:277-283
12.章如安,杨晟,邱荣德,袁中一.巴斯德毕赤酵母表达体系研究及进展[J].微生物学通报,2000,27(5):371-373
13.李健仔.巴斯德毕赤酵母外源基因表达系统[J].微生物学通报,2005,40(3):21-23
14 .龙晶,杜立新.巴斯德毕赤酵母表达外源蛋白的研究进展[J].微生物学杂志,2007,27(6):72-76
15.胡世界,罗素兰,张吉贞,长孙东亭.巴斯德毕赤酵母表达系统及其高水平表达策略[J].生物技术,2007,17(6):78-83
16.戚楠,马清钧.长效重组蛋白药物的研究进展[J].中国生物工程杂志,2006,26(2):79-82
17.王秀贞,吴军,孟宪军.长效多肽药物研究进展[J].中国生物工程杂志,2003,23(10):23-27
18.Lucas L H,Price K E,Larive C K.Epitope mapping and competitive binding of HSA drug site II ligands by NMR diffusion measurements.J Am Chem Soc,2004,126 (43):14258-14266
19.Petes T P. All About Albumin.San Diego:Academic Press,1996
20.Chuang V T,Kragh-Hansen U,Otagiri M. Pharmaceutical strategies utilizing recombinant human serum albumin.PharmRes,2002,19(5):569-577
21.Sung C,Nardelli B,LaFleur D W,et al.An IFN-beta-albumin fusion protein that displays improved pharmacokinetic and pharmacodynamic properties in nonhuman primates.Interferon Cytokine Res,2003,23(1):25-36
22.J Lee G K. PEG conjugation moderately protects adeno-associated viral vectors against antibody neutralization.Biotechnol Bioeng,2005,92(1):24-34
23.Veronese F M,Pasut G. PEGylation,successful approach to drug delivery.Drug Discov Today,2005,10(21):1451-1458
24 . Graham M L. Pegaspargase:a review of clinical studies.Adv Drug Deliv Rev,2003,55(10):1293-1302
25.Michallet M,Maloisel F,Delain M,et al.Pegylated recombinant interferon alpha-2b vs recombinant interfere on alpha-2b for the initial treatment of chronic-phase chronic myelogenous leukemia: a phase III study.Leukemia,2004,18(2):309-315
26 . Molineux G.The design and development of pegfilgrastim(PEG-rmetHuG-CSF, Neulasta).Curr Pharm Des,2004,10(11):1235-1244
27.王秀贞,吴军,孟宪军.长效多肽药物研究进展[J].中国生物工程杂志,2003,23(10):23-27
28.吴诚,钟延强.干扰素缓释给药系统的研究进展[J].药学实践杂志,2003,21(2):87-89
29.罗东.干扰素剂型研究进展[J].中国新医药,2003,2(9):49-51
30.张华金,李娜,杜学志.干扰素的剂型及临床应用[J].华西药学杂志,2004,19(1):53-55
31.Cregg J. M,Barringer K. J,Hessler A.Y,et al.Pichia pastoris as a host system for transformation.Mol CellBiol,1985,5(12):3376-3385
32 . MartineM,Zeng S,Berger E. G.The yeast expression system for recombinant glycosyltransferases.Glycoconjugate Journal,1999,(16):125-139
33.韩雪清,刘湘涛,尹双辉.毕赤酵母表达系统[J].微生物学杂志,2003,33(4):35-40
34.Martínez-Ruiz A,Martínez del Pozo A,Lacadena J,et al.Secretion of recombinant proand mature fungal-sarcin ribotoxin by the methylotropic yeast Pichia pastoris: the Lys-Arg motif is required formaturation.Protein Expr Purif,1998,(12):315-322
35 . Sreekrishna K,Brankamp RG,Kropp KE,et al.Strategies for optimal synthesis of heterologous proteins in the methylotrophic yeast Pichia pastoris. Gene,1997,190:55-62
36.Emma D,Thorpe, Marc C,et al.Sorbitol as a non-repression carbon source for fed-batch fermentation of recombinant pichia pastoris.Biotechnology Letters,1999,21:669-672
37.Wenhui W,Mark AB,Bradly AP,et al.Meling pichia pastoris growth on methanol and optimizing the production of a recombinant protein,the Heavy-chain fragment C of Botulinum neurotoxin Serotype A.Bioengineering,2000,20(1):1-8
38.Hong F,Meinander NQ,Jonsson LJ.Fermentation strategies for improved heterologous expression of laccase in Pichia pastoris.Biotechnol Bioeng,2002,79(4):438-449
39.Kaoru Kobayashi,Shinobu Kuwae,Tomoshi Ohya,et al.Addition of oleic acidincreases expression of recombinant human serum albumin by the AOX2 promoter in Pichia pastoris.Journal of Bioscience and Bioengineering,2000,89(5):479-484
40.Clare JJ,Rayment FB,Ballantyne SP,Sreerkrishna K,Romanos MA.High-level expression of tetanus toxin fragment C in Pichia pastoris strains containing multiple tandem integrations of the gene.BioTechnology,1991,9:455–460
41.邹敏,方曙光,黄立,庄英萍,储炬,张嗣良,严成钊.重组毕赤酵母表达瑞替普酶(Reteplase)过程中的降解控制[J].工业微生物,2006,36(2):7-11
42.Sreektishna K,Brankamp R G,Kropp K E et al. Strategy for optimal synthesis and secretion of heterogulous proteins in the mythy trophic yeast Pichia pastoris.Gene,1997,191(1):55-62
43.MEI M,WHITTAKER,JAMESW,et al.Expression of recombinant galactose oxidase by Pichia pastoris.Protein Expr,Purif,2000,20:105-111
44.Shi X,Karkut T,Chamankhah M,et al.Optimal conditions for the expression of a single-chain antibody (scFv) gene in Pichia pastoris.Prot Exp Pur,2003,28:321–330
45.雷楗勇.人β干扰素-血清白蛋白融合蛋白在毕赤酵母中的分泌表达[J].中国生物工程杂志,2006,26(7):13-18
46.刘永东,王云山,苏志国.重组人血清白蛋白生产工艺研究进展.微生物学通报,2003,30(5):128-132
47.罗昭锋,瞿鑫,沐万孟,时青,张毅.超声和高压处理对牛血清白蛋白结构的影响[J].中国生物工程杂志,2006,26(1):46-49
48.Kobayashi K, Kuwae S, Ohya T, Ohda T, Ohyama M, Ohi H, Tomomitsu K, Ohmura T. 2000. High-level expression of recombinant human serum albumin from the methylotrophic yeast Pichia pastoris with minimal protease production and activation. J Biosci Bioeng 89:55-61.
49.姜微波.凝胶电泳分析蛋白酶活性的技术改进[J].植物学通报,2002,19(5):607-610
50.刘清岱,刘竹芸,任岩,朱晔荣,王勇.检测蛋白酶活性的双向凝胶电泳技术[J].植物生理学通讯,2005,41(6):810-812
51 . Jayanta Sinha,Bradley A. Plantz,Mehmet Inan,Michael M.Meagher.Causes of Proteolytic Degradation of Secreted Recombinant Proteins Produced in Methylotrophic Yeast Pichia pastoris: Case Study With Recombinant Ovine Interferon-τ.Biotechnol Bioeng,2004,89:102-112
52.Gimenez JA,Monkovic DD,Dekleva ML.Identification and monitoring of protease activity in recombinant Saccharomyces cerevisiae.Biotechnol Bioeng,2000,67:245-251
53.卢士英,任洪林,柳增善,赵广英,华育平.组织蛋白酶B研究进展[J].河北师范大学学报,2004,28(3):306-309
54.Sh i X,Karkut T,Chamankhah M,et al.Optimal conditions for the expression of a single-chain antibody (scFv) gene in P.pastoris.Protein Expr Purif,2003,28(2):321-330
55.Van Den Hazel HB,Kiellanbrandt MC,Winther JR.Biosynthesis and function of yeast vacuolar proteases:review.Yeast,1996,12:1-16
56.Hansen RJ,Switzer RL,Hinze H,Holzer H.Effects of glucose and nitrogen source on the levels of proteinases,peptidases,and proteinase inhibitors in yeast.Biochim Biophys Acta. 1977,496:103–114
57.胡成旭,侯欣彤,冯永宁等.响应面法优化云芝多糖提取条件的研究[J].食品工业科技,2007,28(7):124-126
58 .张晓伟,张培正,宫玮等.酶法提取黄蔑多糖的工艺优化[J].食品与发酵工业,2007,33(5):158-161
2.Daly Rachel,Hearn M T W.Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and production.Journal of Molecular Recognition,2005,18:119-138
3.David R.Higgins,JamesM Cremes.Pichia Protocols.New Jersey:Human Press Inc,1998
4.Macauley-Patrick S,FazendaML,McNeil B,et al.Heterologous protein production using the Pichia pastoris expression system.Yeast,2005,22:249-270
5.Cereghino JL,Cregg JM.Heterlogous protein expression in the methylotrophic yeast Pichia pastoris.FEMS Microbiology Reviews,2000,24:45-66
6.Daly R,Hearn MT.Expression of heterologous proteins in Pichia pastoris a useful experimental tool in protein engineering and production.Journal of molecular recognition,2005,18(2):119-138
7.Kout P,Davis,GR,et al.Structural comparison of the Pichia pastoris alcohol oxidase genes.Yeast,1999,5:167-177
8.Thill,G.P.,Davis,G.R., et al.Positive and negative ejects of multicopy integrated expression vectors on protein expression in Pichia pastoris.Proceedings of the Sixth International Symposium on the Genetics of Microorganisms. Socie¨te¨Franc°aise de Microbiologie,1990,2:477-490
9.Brierley,R.A.Secretion of recombinant human insulin-like growth factor I (IGF-1).Methods Mol.Biol.1998,103:149-177
10 . Cregg,J.M. and Madden,K.R.Development of yeast transformation systems and construction of methanol-utilization-defective mutants of Pichia pastoris by gene disruption.Biological Research on Industrial Yeasts(Stewart, G.G., Russell, I., Klein, R.D.and Hiebsch,R.R.,Eds.).1987,2:1-18
11.Chiruvolu,V.,Cregg,J.M. and Meagher,M.M.Recombinant protein production in an alcohol oxidase-defective strain of Pichia pastoris in fed-batch fermentations.Enzyme Microb.Technol.1997,21:277-283
12.章如安,杨晟,邱荣德,袁中一.巴斯德毕赤酵母表达体系研究及进展[J].微生物学通报,2000,27(5):371-373
13.李健仔.巴斯德毕赤酵母外源基因表达系统[J].微生物学通报,2005,40(3):21-23
14 .龙晶,杜立新.巴斯德毕赤酵母表达外源蛋白的研究进展[J].微生物学杂志,2007,27(6):72-76
15.胡世界,罗素兰,张吉贞,长孙东亭.巴斯德毕赤酵母表达系统及其高水平表达策略[J].生物技术,2007,17(6):78-83
16.戚楠,马清钧.长效重组蛋白药物的研究进展[J].中国生物工程杂志,2006,26(2):79-82
17.王秀贞,吴军,孟宪军.长效多肽药物研究进展[J].中国生物工程杂志,2003,23(10):23-27
18.Lucas L H,Price K E,Larive C K.Epitope mapping and competitive binding of HSA drug site II ligands by NMR diffusion measurements.J Am Chem Soc,2004,126 (43):14258-14266
19.Petes T P. All About Albumin.San Diego:Academic Press,1996
20.Chuang V T,Kragh-Hansen U,Otagiri M. Pharmaceutical strategies utilizing recombinant human serum albumin.PharmRes,2002,19(5):569-577
21.Sung C,Nardelli B,LaFleur D W,et al.An IFN-beta-albumin fusion protein that displays improved pharmacokinetic and pharmacodynamic properties in nonhuman primates.Interferon Cytokine Res,2003,23(1):25-36
22.J Lee G K. PEG conjugation moderately protects adeno-associated viral vectors against antibody neutralization.Biotechnol Bioeng,2005,92(1):24-34
23.Veronese F M,Pasut G. PEGylation,successful approach to drug delivery.Drug Discov Today,2005,10(21):1451-1458
24 . Graham M L. Pegaspargase:a review of clinical studies.Adv Drug Deliv Rev,2003,55(10):1293-1302
25.Michallet M,Maloisel F,Delain M,et al.Pegylated recombinant interferon alpha-2b vs recombinant interfere on alpha-2b for the initial treatment of chronic-phase chronic myelogenous leukemia: a phase III study.Leukemia,2004,18(2):309-315
26 . Molineux G.The design and development of pegfilgrastim(PEG-rmetHuG-CSF, Neulasta).Curr Pharm Des,2004,10(11):1235-1244
27.王秀贞,吴军,孟宪军.长效多肽药物研究进展[J].中国生物工程杂志,2003,23(10):23-27
28.吴诚,钟延强.干扰素缓释给药系统的研究进展[J].药学实践杂志,2003,21(2):87-89
29.罗东.干扰素剂型研究进展[J].中国新医药,2003,2(9):49-51
30.张华金,李娜,杜学志.干扰素的剂型及临床应用[J].华西药学杂志,2004,19(1):53-55
31.Cregg J. M,Barringer K. J,Hessler A.Y,et al.Pichia pastoris as a host system for transformation.Mol CellBiol,1985,5(12):3376-3385
32 . MartineM,Zeng S,Berger E. G.The yeast expression system for recombinant glycosyltransferases.Glycoconjugate Journal,1999,(16):125-139
33.韩雪清,刘湘涛,尹双辉.毕赤酵母表达系统[J].微生物学杂志,2003,33(4):35-40
34.Martínez-Ruiz A,Martínez del Pozo A,Lacadena J,et al.Secretion of recombinant proand mature fungal-sarcin ribotoxin by the methylotropic yeast Pichia pastoris: the Lys-Arg motif is required formaturation.Protein Expr Purif,1998,(12):315-322
35 . Sreekrishna K,Brankamp RG,Kropp KE,et al.Strategies for optimal synthesis of heterologous proteins in the methylotrophic yeast Pichia pastoris. Gene,1997,190:55-62
36.Emma D,Thorpe, Marc C,et al.Sorbitol as a non-repression carbon source for fed-batch fermentation of recombinant pichia pastoris.Biotechnology Letters,1999,21:669-672
37.Wenhui W,Mark AB,Bradly AP,et al.Meling pichia pastoris growth on methanol and optimizing the production of a recombinant protein,the Heavy-chain fragment C of Botulinum neurotoxin Serotype A.Bioengineering,2000,20(1):1-8
38.Hong F,Meinander NQ,Jonsson LJ.Fermentation strategies for improved heterologous expression of laccase in Pichia pastoris.Biotechnol Bioeng,2002,79(4):438-449
39.Kaoru Kobayashi,Shinobu Kuwae,Tomoshi Ohya,et al.Addition of oleic acidincreases expression of recombinant human serum albumin by the AOX2 promoter in Pichia pastoris.Journal of Bioscience and Bioengineering,2000,89(5):479-484
40.Clare JJ,Rayment FB,Ballantyne SP,Sreerkrishna K,Romanos MA.High-level expression of tetanus toxin fragment C in Pichia pastoris strains containing multiple tandem integrations of the gene.BioTechnology,1991,9:455–460
41.邹敏,方曙光,黄立,庄英萍,储炬,张嗣良,严成钊.重组毕赤酵母表达瑞替普酶(Reteplase)过程中的降解控制[J].工业微生物,2006,36(2):7-11
42.Sreektishna K,Brankamp R G,Kropp K E et al. Strategy for optimal synthesis and secretion of heterogulous proteins in the mythy trophic yeast Pichia pastoris.Gene,1997,191(1):55-62
43.MEI M,WHITTAKER,JAMESW,et al.Expression of recombinant galactose oxidase by Pichia pastoris.Protein Expr,Purif,2000,20:105-111
44.Shi X,Karkut T,Chamankhah M,et al.Optimal conditions for the expression of a single-chain antibody (scFv) gene in Pichia pastoris.Prot Exp Pur,2003,28:321–330
45.雷楗勇.人β干扰素-血清白蛋白融合蛋白在毕赤酵母中的分泌表达[J].中国生物工程杂志,2006,26(7):13-18
46.刘永东,王云山,苏志国.重组人血清白蛋白生产工艺研究进展.微生物学通报,2003,30(5):128-132
47.罗昭锋,瞿鑫,沐万孟,时青,张毅.超声和高压处理对牛血清白蛋白结构的影响[J].中国生物工程杂志,2006,26(1):46-49
48.Kobayashi K, Kuwae S, Ohya T, Ohda T, Ohyama M, Ohi H, Tomomitsu K, Ohmura T. 2000. High-level expression of recombinant human serum albumin from the methylotrophic yeast Pichia pastoris with minimal protease production and activation. J Biosci Bioeng 89:55-61.
49.姜微波.凝胶电泳分析蛋白酶活性的技术改进[J].植物学通报,2002,19(5):607-610
50.刘清岱,刘竹芸,任岩,朱晔荣,王勇.检测蛋白酶活性的双向凝胶电泳技术[J].植物生理学通讯,2005,41(6):810-812
51 . Jayanta Sinha,Bradley A. Plantz,Mehmet Inan,Michael M.Meagher.Causes of Proteolytic Degradation of Secreted Recombinant Proteins Produced in Methylotrophic Yeast Pichia pastoris: Case Study With Recombinant Ovine Interferon-τ.Biotechnol Bioeng,2004,89:102-112
52.Gimenez JA,Monkovic DD,Dekleva ML.Identification and monitoring of protease activity in recombinant Saccharomyces cerevisiae.Biotechnol Bioeng,2000,67:245-251
53.卢士英,任洪林,柳增善,赵广英,华育平.组织蛋白酶B研究进展[J].河北师范大学学报,2004,28(3):306-309
54.Sh i X,Karkut T,Chamankhah M,et al.Optimal conditions for the expression of a single-chain antibody (scFv) gene in P.pastoris.Protein Expr Purif,2003,28(2):321-330
55.Van Den Hazel HB,Kiellanbrandt MC,Winther JR.Biosynthesis and function of yeast vacuolar proteases:review.Yeast,1996,12:1-16
56.Hansen RJ,Switzer RL,Hinze H,Holzer H.Effects of glucose and nitrogen source on the levels of proteinases,peptidases,and proteinase inhibitors in yeast.Biochim Biophys Acta. 1977,496:103–114
57.胡成旭,侯欣彤,冯永宁等.响应面法优化云芝多糖提取条件的研究[J].食品工业科技,2007,28(7):124-126
58 .张晓伟,张培正,宫玮等.酶法提取黄蔑多糖的工艺优化[J].食品与发酵工业,2007,33(5):158-161