重组蛋白GGH在毕赤酵母中高效表达及纯化
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摘要
GLP-1是由肠道L细胞合成和分泌的由30个氨基酸组成的多肽,它不仅能促进胰岛素合成,而且能够促进胰β细胞增殖和抑制胰β细胞凋亡。GLP-1独特的降血糖作用机制,是现有抗糖尿病药物无可比拟的,但由于其在体内的半衰期非常短,大大限制了其应用。为克服GLP-1半衰期短的缺点,实现其在体内的长效作用,本实验室成功构建了胰高糖素样多肽-1(GLP-1)突变串联体与人血清白蛋白的表达质粒pPIC9K/(GLP-1A2G)2-HSA,并且初步实现了在毕赤酵母KM71中的表达。在此基础上,本文主要围绕提高融合蛋白GGH的产量和分离纯化进行研究,主要结论如下:
(1)统计分析了融合基因GGH在不同毕赤酵母宿主(KM71、GS115、SMD1168)中的表达,并通过荧光定量PCR研究了三株产量差异较大的GS115重组子基因剂量与融合蛋白GGH表达量的关系,实验结果表明:宿主GS115更有利于融合蛋白GGH的表达,融合蛋白的表达量与目的基因的剂量成正相关,最终确定高一株产菌株GS115/F2,在摇甁中的稳定表达量为328 mg/L。
(2)研究了高表达菌株的发酵过程,确定了其在5 L发酵罐上的发酵工艺。实验结果表明有机氮源培养基更有利于融合蛋白GGH高密度表达及控制目的蛋白的降解;在有机氮源培养基中,0.5%的甲醇脉冲式流加方式和30℃诱导温度为最佳诱导条件,最终融合蛋白GGH的产量达到642 mg/L。
(3)初步建立了融合蛋白GGH的分离纯化路线,即发酵液8000 r/min离心5 min, 10 kDa截留量的超滤膜超滤浓缩20倍, Blue Sepharose亲和吸附、Sephadex G25脱盐、Q Sepharose FF离子交换层析,整个纯化过程回收率为33.4%,经SDS-PAGE和HPLC检测纯度大于95%,符合下一步生物活性和药代动力学实验和药剂学试验的要求。
Glucagon-like peptide-1(GLP-1) is 30-residual peptide hormone secreted by intestinal L-cells in response to nutrient ingestion. It plays an important role in simulating insulin secretion, inducing pancreatic beta cells proliferation and inhibiting pancreatic beta cells apoptosis. The unique hypoglycemic mechanism of GLP-1 is unparalleled to the existing anti-diabetes drugs. Consequence to its natural function as locally secreted short-term messenger, its half-life in circulation is very short and provided mainly by fast renal clearance due to its low molecular weight and proteolysis susceptibility, resulting in low clinical utility. In our lab, A GLP-1 fusion protein named GGH comprising double tandem GLP-1 and human serum albumin (HSA) was constructed to prolong its half-life. The GLP-1 fusion protein was expressed in pichia pastoris KM71 transformed with vector pPIC9K/(GLP-1A2G)2-HSA. In this study, improving the expression level and purification of fusion protein GGH were focused on. The main conclusions are as follows:
The fusion protein GGH expression level in different host strains (KM71, GS115 and SMD1168) was determined and analyzed by statistical methods. The relevance of gene dosage and fusion protein GGH expression level was investigated by RT-PCR in three GS115 recombinants with different expression level. The results showed that the host strain GS115 was more suitable for expressing fusion protein GGH. It was a positive correlation between gene dosage and fusion protein GGH expression level. A high yield recombinant strain GS115/F2 with expression level of 328 mg/L in shake flask culture was selected out for further study.
After investigating the fermentation process of high yield recombinant strain, the process was confirmed. The results demonstrated that organic nitrogen sources were more conducive to high density culture of recombinant strain and inhibiting proteolysis. A pulse feeding strategy of 0.5% methanol each time was the optimal mode for inducing fusion protein expression. 30℃was the optimal temperature for inducing. The yield of fusion protein GGH in the fermentation process of recombinant strain reached to 642 mg/L。
A preliminary purification process was established. The process with a total recovery of 33.4% included five steps: centrifugation, filtration, Blue sepharose, Sephadex G25 and Q sepharose chromatography. The fusion protein GGH was purified to apparent homogeneity with purity greater than 95% analyzed by SDS-PAGE and HPLC, meeting the requirement of the study of pharmacodynamics, pharmacokinetics and pharmacy.
(1)统计分析了融合基因GGH在不同毕赤酵母宿主(KM71、GS115、SMD1168)中的表达,并通过荧光定量PCR研究了三株产量差异较大的GS115重组子基因剂量与融合蛋白GGH表达量的关系,实验结果表明:宿主GS115更有利于融合蛋白GGH的表达,融合蛋白的表达量与目的基因的剂量成正相关,最终确定高一株产菌株GS115/F2,在摇甁中的稳定表达量为328 mg/L。
(2)研究了高表达菌株的发酵过程,确定了其在5 L发酵罐上的发酵工艺。实验结果表明有机氮源培养基更有利于融合蛋白GGH高密度表达及控制目的蛋白的降解;在有机氮源培养基中,0.5%的甲醇脉冲式流加方式和30℃诱导温度为最佳诱导条件,最终融合蛋白GGH的产量达到642 mg/L。
(3)初步建立了融合蛋白GGH的分离纯化路线,即发酵液8000 r/min离心5 min, 10 kDa截留量的超滤膜超滤浓缩20倍, Blue Sepharose亲和吸附、Sephadex G25脱盐、Q Sepharose FF离子交换层析,整个纯化过程回收率为33.4%,经SDS-PAGE和HPLC检测纯度大于95%,符合下一步生物活性和药代动力学实验和药剂学试验的要求。
Glucagon-like peptide-1(GLP-1) is 30-residual peptide hormone secreted by intestinal L-cells in response to nutrient ingestion. It plays an important role in simulating insulin secretion, inducing pancreatic beta cells proliferation and inhibiting pancreatic beta cells apoptosis. The unique hypoglycemic mechanism of GLP-1 is unparalleled to the existing anti-diabetes drugs. Consequence to its natural function as locally secreted short-term messenger, its half-life in circulation is very short and provided mainly by fast renal clearance due to its low molecular weight and proteolysis susceptibility, resulting in low clinical utility. In our lab, A GLP-1 fusion protein named GGH comprising double tandem GLP-1 and human serum albumin (HSA) was constructed to prolong its half-life. The GLP-1 fusion protein was expressed in pichia pastoris KM71 transformed with vector pPIC9K/(GLP-1A2G)2-HSA. In this study, improving the expression level and purification of fusion protein GGH were focused on. The main conclusions are as follows:
The fusion protein GGH expression level in different host strains (KM71, GS115 and SMD1168) was determined and analyzed by statistical methods. The relevance of gene dosage and fusion protein GGH expression level was investigated by RT-PCR in three GS115 recombinants with different expression level. The results showed that the host strain GS115 was more suitable for expressing fusion protein GGH. It was a positive correlation between gene dosage and fusion protein GGH expression level. A high yield recombinant strain GS115/F2 with expression level of 328 mg/L in shake flask culture was selected out for further study.
After investigating the fermentation process of high yield recombinant strain, the process was confirmed. The results demonstrated that organic nitrogen sources were more conducive to high density culture of recombinant strain and inhibiting proteolysis. A pulse feeding strategy of 0.5% methanol each time was the optimal mode for inducing fusion protein expression. 30℃was the optimal temperature for inducing. The yield of fusion protein GGH in the fermentation process of recombinant strain reached to 642 mg/L。
A preliminary purification process was established. The process with a total recovery of 33.4% included five steps: centrifugation, filtration, Blue sepharose, Sephadex G25 and Q sepharose chromatography. The fusion protein GGH was purified to apparent homogeneity with purity greater than 95% analyzed by SDS-PAGE and HPLC, meeting the requirement of the study of pharmacodynamics, pharmacokinetics and pharmacy.
引文
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4. Orskov C,Rabenh?j L,Wettergren A,Kofod H,Holst JJ.Tissue and plasma concentrations of amidated and glycine-extended glucagon-like peptide I in humans[J]. 1994,43(4):535-539.
5. Orskov C,Wettergren A,Holst JJ. Biological effects and metabolic rates of glucagonlike peptide-1 7-36 amide and glucagonlike peptide-1 7-37 in healthy subjects are indistinguishable[J]. Diabetes, 1993,42(5):658-661.
6. Adrian V,Pankaj S,Rita B,et a1. Efect of glucagom-like peptide-1(7-36)amide on initial splanchnic gIucose uptake and insulin action in humans with type 1 diabetes[J]. Diabetes,2001,50:565.
7. Langtly H D,Balfour J A,Langtry H D,et al. Glimepiride. A review of its use in the management of type 2 diabetes mellitue[J]. Drugs,1998,55 (4):563-571.
8. Turton M D,O'Shea D,Gunn I,et al. A role for glucagon-like peptide-1 in the central regulation of feeding[J]. Nature,1996,379(6560):69-72.
9. Deacon C F,Johnsen A H,Holst J J,Degradation of glucagon-like peptide-1 by human plasma in vitro yields an N-terminally truncated peptide that is a major endogenous metabolite in vivo[J]. J. Clin. Endocrinol. Metab, 1995,80:952-957.
10.张志珍,刘智慧.人胰高血糖素样肽-1突变体基因的克隆及表达[J].生物技术, 2004,14(3):4-7.
11. Zhihui GAO,Gang BAI,Jiaqi CHEN et a1.Development,Characterization,and Evaluaton of a fusion proteion of noval glucagon-like peptide-1(GLP-1) analog and human serum albumin in Pichia pastoris[J]. Biosic.Biotechnol.Biochem,2009,73(3):688-694.
12. Lee S H,L ee S,Youn Y S,et al.Synthesis,characterization,and pharmacok inetic studies of PEGylated glucagons-like pep tide-1[J]. Bioconjug Chem,2005,16(2):377.
13.葛晓宇,刘景晶. Exendin-4及其类似物的研究近况[J]. 2007,31(9):403-407.
14. Petes T P. All About Albumin[M]. San Diego: Academic Press,1996.
15.殷艺伟,陈建华.重组人血清白蛋白的药学应用研究进展[J].药学进展,2006,30(2):70-74.
16. Kobayashi K,Nakamura N,Sumi A,et al.The development of recombinant human serum albumin. Ther Apher. 1998,2(4):257-262.
17. Osborn B L, Olsen H S,Nardelli B, et al. Pharmacokinetic and pharmacodynamic studies of a human serum albumin interferon-2alpha fusion p rotein in cynomolgus monkeys. J Pharmacol Exp Ther. 2002,303(2):540～548.
18.龙娟,薛冲,刘志敏.人胰高血糖素样肽-1与人血清白蛋白融合蛋白在毕赤酵母中的表达[J].生物技术通讯. 2008,15(4):488-493.
19.孙纯义,李元,李别虎.在毕赤酵母菌中表达的hGlp-1-白蛋白融合蛋白的纯化[J].大连民族学院学报. 2007,40(5):76-79.
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21. Cregg J M, Cereghino L,Shi J,Higgins DR: Recombinant protein expression in Pichia pastoris. Mol Biotech. 2000,16:23-52.
22. Pau Ferrer,Francisco Valero, Sorbitol co-feeding reduces metabolic burden caused by the overexpression of a Rhizopus oryzae lipase in Pichia pastoris. Bioconjug Chem. 2007,16(2):377.
23. Sreekrishna K,Brankamp R G,Kropp K E,et al. Strategies for optimal synthesis of heterologous proteins in the methylotrophic yeast Pichia pastoris [J]. Gene. 1997,190: 55-62.
24. Hauhan A.K.,Arora D,Khanna N.A. Novel Feeding Strategy for Enhanced Protein Production by Fed-batch Fermentation in Recombinant Pichia pastoris[J]. Process Biochemistry, 1999,34(3):139-145.
25. Carmen J,Ian M,Urs S. Mixed feeds of glycerol and methanol can improve the performance of Pichia pastoris cultures: A quantitative study based on concentration gradients in transient continuous cultures Journal of Biotechnology[J]. 2007,128(4):824-837.
26.孙彦.生物分离工程[M].北京:化学工业出版社,1998.111-124.
27.袁辉.药物蛋白质分离纯化技术[M].北京:化学工业出版社,2003.55-72.
28.顾觉奋,李丽燕,刘叶青.分离纯化工艺原理[M].北京:中国医药科技出版社,1996.21-49.
29. Hasslacher M, Schall M. High-level intracelullar expression of hydroxynitrile lyase from the tropical rubber tree Hevea brasiliensis in microbial hosts[J]. Protein Expr Purif, 1997,11:61.
30. Hohenblum H,Gasser B,Maurer M.et a1.Effects of gene dosage,promoters and substrates on unfolded protein stress of recombinant Pichia pastoris. Biotechnol Bioeng. 2004,85(4):367-375.
31. Sreekrishna K,Nelles L,Potenz R,et a1.High—level expression,purification,and characterization of recombinant human tumor necrosis factor synthesized in the methylotrophic yeast Pichia pastoris. Biochemistry. 1989,28(9):4117-4125.
32. Clare J J,Rayment F B,Ballantyne S P,Sreerkrishna K,Romanos M A. High-level expression of tetanus toxin fragment C in Pichia pastoris strains containing multiple tandem integrations of the gene. BioTechnology. 1991,9:455–460.
33. Clare J J,Romanos M A,Rayment F B,et a1.Production of mouse epidermal growth factor in yeast: high-level secretion using Pichia pastoris strains containing mutiple gene copies. Gene. 1991,105:205-212.
34. Vassileva A,Chugh D A,Swaminathan S,et a1.Efect of copy number on the expression levels of hepatitis B suIface antigen in the methylotrophic yeast Pichia pastoris. Protein Expression and Purification. 2001,21(1):71-80.
35. Wagner S L,Siegel R S,Vedvick T S,et a1.High—level expression,purification and characterization of Kunitz-type protease inhibitor domain of protease nexin-2/amyloid-beta -protein precursor. Biochem Biophys Res Commun. 1992,186:1138-114.
36. Cos O , Serrano A , Montesinos J L , et a1. Combined efect of the methanol utilization(Mut)phenotype and gene dosage on recombinant protein production in Pichia pastoris fed—batch cultures. J Biotechnol. 2005,116(4):321-335.
37. Cereghino, J L. and Cregg, J M. Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol. Rev, 2000,24:45-66.
38. Cregg J M, Madden K R. Development of the methylotrophic yeast,Pichia pastoris, as a host system for the production of foreign proteins. Dev Ind Microbiol. 1988,29:23-42.
39. Klaus W (ed) . Nonconventional yeasts in biotechnology,Berlin,Germany,Springer. 1996,203-253.
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2. Drucker D J. Minireview: the glucagon-like peptides[J]. Endocrinology, 2001,142(2): 521-527.
3. Zander M,Madsbad S,Madsen J L,et al. Effect of 6-week course of glucagon-like peptide 1 on glycaemic control,insulin sensitivity,and beta-cell function in type 2 diabetes: a parallel-group study[J].Lancet, 2002,359(9003):824-830.
4. Orskov C,Rabenh?j L,Wettergren A,Kofod H,Holst JJ.Tissue and plasma concentrations of amidated and glycine-extended glucagon-like peptide I in humans[J]. 1994,43(4):535-539.
5. Orskov C,Wettergren A,Holst JJ. Biological effects and metabolic rates of glucagonlike peptide-1 7-36 amide and glucagonlike peptide-1 7-37 in healthy subjects are indistinguishable[J]. Diabetes, 1993,42(5):658-661.
6. Adrian V,Pankaj S,Rita B,et a1. Efect of glucagom-like peptide-1(7-36)amide on initial splanchnic gIucose uptake and insulin action in humans with type 1 diabetes[J]. Diabetes,2001,50:565.
7. Langtly H D,Balfour J A,Langtry H D,et al. Glimepiride. A review of its use in the management of type 2 diabetes mellitue[J]. Drugs,1998,55 (4):563-571.
8. Turton M D,O'Shea D,Gunn I,et al. A role for glucagon-like peptide-1 in the central regulation of feeding[J]. Nature,1996,379(6560):69-72.
9. Deacon C F,Johnsen A H,Holst J J,Degradation of glucagon-like peptide-1 by human plasma in vitro yields an N-terminally truncated peptide that is a major endogenous metabolite in vivo[J]. J. Clin. Endocrinol. Metab, 1995,80:952-957.
10.张志珍,刘智慧.人胰高血糖素样肽-1突变体基因的克隆及表达[J].生物技术, 2004,14(3):4-7.
11. Zhihui GAO,Gang BAI,Jiaqi CHEN et a1.Development,Characterization,and Evaluaton of a fusion proteion of noval glucagon-like peptide-1(GLP-1) analog and human serum albumin in Pichia pastoris[J]. Biosic.Biotechnol.Biochem,2009,73(3):688-694.
12. Lee S H,L ee S,Youn Y S,et al.Synthesis,characterization,and pharmacok inetic studies of PEGylated glucagons-like pep tide-1[J]. Bioconjug Chem,2005,16(2):377.
13.葛晓宇,刘景晶. Exendin-4及其类似物的研究近况[J]. 2007,31(9):403-407.
14. Petes T P. All About Albumin[M]. San Diego: Academic Press,1996.
15.殷艺伟,陈建华.重组人血清白蛋白的药学应用研究进展[J].药学进展,2006,30(2):70-74.
16. Kobayashi K,Nakamura N,Sumi A,et al.The development of recombinant human serum albumin. Ther Apher. 1998,2(4):257-262.
17. Osborn B L, Olsen H S,Nardelli B, et al. Pharmacokinetic and pharmacodynamic studies of a human serum albumin interferon-2alpha fusion p rotein in cynomolgus monkeys. J Pharmacol Exp Ther. 2002,303(2):540～548.
18.龙娟,薛冲,刘志敏.人胰高血糖素样肽-1与人血清白蛋白融合蛋白在毕赤酵母中的表达[J].生物技术通讯. 2008,15(4):488-493.
19.孙纯义,李元,李别虎.在毕赤酵母菌中表达的hGlp-1-白蛋白融合蛋白的纯化[J].大连民族学院学报. 2007,40(5):76-79.
20. Cregg,J.M. and Madden, K.R. Development of the methylotrophic yeast,Pichia pastoris, as a host system for the production of foreign proteins. Dev. Ind. Microbiol. 1988,29:33-41.
21. Cregg J M, Cereghino L,Shi J,Higgins DR: Recombinant protein expression in Pichia pastoris. Mol Biotech. 2000,16:23-52.
22. Pau Ferrer,Francisco Valero, Sorbitol co-feeding reduces metabolic burden caused by the overexpression of a Rhizopus oryzae lipase in Pichia pastoris. Bioconjug Chem. 2007,16(2):377.
23. Sreekrishna K,Brankamp R G,Kropp K E,et al. Strategies for optimal synthesis of heterologous proteins in the methylotrophic yeast Pichia pastoris [J]. Gene. 1997,190: 55-62.
24. Hauhan A.K.,Arora D,Khanna N.A. Novel Feeding Strategy for Enhanced Protein Production by Fed-batch Fermentation in Recombinant Pichia pastoris[J]. Process Biochemistry, 1999,34(3):139-145.
25. Carmen J,Ian M,Urs S. Mixed feeds of glycerol and methanol can improve the performance of Pichia pastoris cultures: A quantitative study based on concentration gradients in transient continuous cultures Journal of Biotechnology[J]. 2007,128(4):824-837.
26.孙彦.生物分离工程[M].北京:化学工业出版社,1998.111-124.
27.袁辉.药物蛋白质分离纯化技术[M].北京:化学工业出版社,2003.55-72.
28.顾觉奋,李丽燕,刘叶青.分离纯化工艺原理[M].北京:中国医药科技出版社,1996.21-49.
29. Hasslacher M, Schall M. High-level intracelullar expression of hydroxynitrile lyase from the tropical rubber tree Hevea brasiliensis in microbial hosts[J]. Protein Expr Purif, 1997,11:61.
30. Hohenblum H,Gasser B,Maurer M.et a1.Effects of gene dosage,promoters and substrates on unfolded protein stress of recombinant Pichia pastoris. Biotechnol Bioeng. 2004,85(4):367-375.
31. Sreekrishna K,Nelles L,Potenz R,et a1.High—level expression,purification,and characterization of recombinant human tumor necrosis factor synthesized in the methylotrophic yeast Pichia pastoris. Biochemistry. 1989,28(9):4117-4125.
32. Clare J J,Rayment F B,Ballantyne S P,Sreerkrishna K,Romanos M A. High-level expression of tetanus toxin fragment C in Pichia pastoris strains containing multiple tandem integrations of the gene. BioTechnology. 1991,9:455–460.
33. Clare J J,Romanos M A,Rayment F B,et a1.Production of mouse epidermal growth factor in yeast: high-level secretion using Pichia pastoris strains containing mutiple gene copies. Gene. 1991,105:205-212.
34. Vassileva A,Chugh D A,Swaminathan S,et a1.Efect of copy number on the expression levels of hepatitis B suIface antigen in the methylotrophic yeast Pichia pastoris. Protein Expression and Purification. 2001,21(1):71-80.
35. Wagner S L,Siegel R S,Vedvick T S,et a1.High—level expression,purification and characterization of Kunitz-type protease inhibitor domain of protease nexin-2/amyloid-beta -protein precursor. Biochem Biophys Res Commun. 1992,186:1138-114.
36. Cos O , Serrano A , Montesinos J L , et a1. Combined efect of the methanol utilization(Mut)phenotype and gene dosage on recombinant protein production in Pichia pastoris fed—batch cultures. J Biotechnol. 2005,116(4):321-335.
37. Cereghino, J L. and Cregg, J M. Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol. Rev, 2000,24:45-66.
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