小分子伴侣的制备及其协助基因工程蛋白体外重折叠的初步研究
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摘要
小分子伴侣GroEL(191-345)是分子伴侣GroEL顶端区域氨基酸残基191-345的片断，它能够显著提高基因工程蛋白如蝎毒Cn5、亲环蛋白A和吲哚3-甘油磷酸合成酶的复性效率，具有广阔的应用前景。为深入研究小分子伴侣协助蛋白质复性的机理，提高包涵体的复性收率，本文将小分子伴侣质粒成功转化后，对小分子伴侣培养条件进行了优化、并将纯化后的小分子伴侣初步应用于重组蛋白的体外复性之中。
首先根据载体的特性，我们选择了大肠杆菌BL21作为宿主细胞。在将质粒导入大肠杆菌后，根据小分子伴侣的分子量18kDa和其六聚组氨酸尾能够亲和吸附到Ni-NTA的特性进行了初步鉴定，并根据小分子伴侣表达量的大小选定了菌种。
然后对培养基的种类、组成和培养条件中各项主要因素进行了考察。在基本培养基的选择时，发现携带小分子伴侣基因的工程菌在含有无机盐和碳源的M9培养基中培养能够显著提高小分子伴侣的表达量，在确定M9为基本培养基之后，对其主要成分进行了优化，同时，考察了发酵温度、供氧量、诱导条件等对目标蛋白表达量的影响，在摇瓶培养中将发酵产量提高到了556.3mg／L。
在转速为6000rpm的条件下收集发酵液中的细胞，用缓冲液将其重新悬浮后，超声波破碎，离心取上清，根据小分子伴侣N末端的His_6-Tag的特性进行了Ni-NTA亲和层析。在层析过程中，对其吸附和洗脱进行了咪唑梯度的考察，实现了一步纯化。纯化后的产品用凝胶层析进行了纯度检测，并用MALDI-TOF-MS验证了纯化后产品的分子量18kDa。
最后以基因工程药物重组人IFN-γ作为底物蛋白，对游离小分子伴侣协助蛋白质复性过程中的主要影响因素进行了初步探讨，结果表明IFN-γ在最终浓度为0.22mg／ml的稀释复性中，加入摩尔量为5倍的游离小分子伴侣，可以使其复性效率提高将近10倍，该方法进而可应用于其它基因工程产物及抗体、疫苗等的复性之中。
The apical domain of GroEL(residues 191-345)was expressed in E.coli to give a functional mini-chaperone, and the refolding yields of scorpion toxin Cn5, Cyclophilin A and IGPS were improved remarkably assisted by mini-chaperone. In order to find its potentially broad application for more proteins, the mechanism of refolding proteins assisted by mini-chaperone need to be further investigated. In this thesis, the plasmid containing the gene of mini-chaperone was successfully transformed into E.coli, the culture condition and expression condition of the transformant was optimized and the purified recombinant mini-chaperone was applied to assist refolding proteins in vitro.
Firstly, according to the nature of the vector, E.coli BL21 was chosen to be the host cell, and the transformation was proved to be a success. Based upon the relative expression amount of mini-chaperone and according to the relative expression amount of mini-chaperone, the No.l strain was selected to be the seed.
Then, the optimum culture condition of mini-chaperone was studied in detail. M9 medium with carbon source and inorganic salts was found to be suitable for the expression of mini-chaperone GroEL(191-345) by E.coli. M9 medium was then reformulated and the optimum temperature, oxygen demand and induction conditions etc. were carefully investigated to improve the mini-chaperone production, the yield of mini-chaperone expressed in Kcoli BL21 reached 556.3 mg/L.
For the mini-chaperone was a fusion protein with a His6-tag at N terminal, it was chosen to be purified by IMAC with the Ni-NTA column. Harvesting the cells by spinning at 6000rpm at 4癈, re-suspending the cells, cracking the cells by sonication, collecting the supernatant by spinning at lOOQOrpm, the sample was loaded for IMAC chromatography. By carefully studied the imidazole concentration in the process of adsorption and elution, pure mini-chaperone was gained by one step. The purity of mini-chaperone gained by IMAC was checked by SEC, and also the molecular weight of mini-chaperone was confirmed to be 18kDa by MALDI-TOF-MS.
Finally, the application of mini-chaperone in refolding recombinant human interferon- y was studied. The main influencing factors of the refolding conditions were investigated, and the refolding yield of IFN-y could improve near 10 folds while adding the 5:1 mini-chaperone to 0.22mg/ml IFN-y in batch. That refolding yield also promised to the further application in other genetically engineered productions.
首先根据载体的特性，我们选择了大肠杆菌BL21作为宿主细胞。在将质粒导入大肠杆菌后，根据小分子伴侣的分子量18kDa和其六聚组氨酸尾能够亲和吸附到Ni-NTA的特性进行了初步鉴定，并根据小分子伴侣表达量的大小选定了菌种。
然后对培养基的种类、组成和培养条件中各项主要因素进行了考察。在基本培养基的选择时，发现携带小分子伴侣基因的工程菌在含有无机盐和碳源的M9培养基中培养能够显著提高小分子伴侣的表达量，在确定M9为基本培养基之后，对其主要成分进行了优化，同时，考察了发酵温度、供氧量、诱导条件等对目标蛋白表达量的影响，在摇瓶培养中将发酵产量提高到了556.3mg／L。
在转速为6000rpm的条件下收集发酵液中的细胞，用缓冲液将其重新悬浮后，超声波破碎，离心取上清，根据小分子伴侣N末端的His_6-Tag的特性进行了Ni-NTA亲和层析。在层析过程中，对其吸附和洗脱进行了咪唑梯度的考察，实现了一步纯化。纯化后的产品用凝胶层析进行了纯度检测，并用MALDI-TOF-MS验证了纯化后产品的分子量18kDa。
最后以基因工程药物重组人IFN-γ作为底物蛋白，对游离小分子伴侣协助蛋白质复性过程中的主要影响因素进行了初步探讨，结果表明IFN-γ在最终浓度为0.22mg／ml的稀释复性中，加入摩尔量为5倍的游离小分子伴侣，可以使其复性效率提高将近10倍，该方法进而可应用于其它基因工程产物及抗体、疫苗等的复性之中。
The apical domain of GroEL(residues 191-345)was expressed in E.coli to give a functional mini-chaperone, and the refolding yields of scorpion toxin Cn5, Cyclophilin A and IGPS were improved remarkably assisted by mini-chaperone. In order to find its potentially broad application for more proteins, the mechanism of refolding proteins assisted by mini-chaperone need to be further investigated. In this thesis, the plasmid containing the gene of mini-chaperone was successfully transformed into E.coli, the culture condition and expression condition of the transformant was optimized and the purified recombinant mini-chaperone was applied to assist refolding proteins in vitro.
Firstly, according to the nature of the vector, E.coli BL21 was chosen to be the host cell, and the transformation was proved to be a success. Based upon the relative expression amount of mini-chaperone and according to the relative expression amount of mini-chaperone, the No.l strain was selected to be the seed.
Then, the optimum culture condition of mini-chaperone was studied in detail. M9 medium with carbon source and inorganic salts was found to be suitable for the expression of mini-chaperone GroEL(191-345) by E.coli. M9 medium was then reformulated and the optimum temperature, oxygen demand and induction conditions etc. were carefully investigated to improve the mini-chaperone production, the yield of mini-chaperone expressed in Kcoli BL21 reached 556.3 mg/L.
For the mini-chaperone was a fusion protein with a His6-tag at N terminal, it was chosen to be purified by IMAC with the Ni-NTA column. Harvesting the cells by spinning at 6000rpm at 4癈, re-suspending the cells, cracking the cells by sonication, collecting the supernatant by spinning at lOOQOrpm, the sample was loaded for IMAC chromatography. By carefully studied the imidazole concentration in the process of adsorption and elution, pure mini-chaperone was gained by one step. The purity of mini-chaperone gained by IMAC was checked by SEC, and also the molecular weight of mini-chaperone was confirmed to be 18kDa by MALDI-TOF-MS.
Finally, the application of mini-chaperone in refolding recombinant human interferon- y was studied. The main influencing factors of the refolding conditions were investigated, and the refolding yield of IFN-y could improve near 10 folds while adding the 5:1 mini-chaperone to 0.22mg/ml IFN-y in batch. That refolding yield also promised to the further application in other genetically engineered productions.
引文
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3) 凌明圣，许祥裕，丁树标．以包涵体形式存在的重组蛋白的纯化和体外折叠．中国生化药物杂志，1995，16(3)，135～139
4) Bowden G A, Georgious G. Folding and Aggregation of β-lactamase in the Periplasmic Space of Escherichia Coli. J. Biol. Chem., 1990, 265, 16760～16766
5) 徐明波，姚志建，原核基因工程中的包涵体．生物化学与生物物理进展，1992，19(2)，89～92
6) Xie Y, Wetlaufer D B. Control of Aggregation in Protein Refolding: the Temperature-leap Tactic. Protein Sci, 1996, 5(3): 571～523
7) Georgious G, Valax P. Expression of Correctly Folded Proteins in E. coli. Curr. Opin. Biotechnol., 1996, 7(2): 190～197
8) 杨云贵，童芹，郑卫东等．分子伴侣过量表达对蛋白质分泌及可溶性的影响．中国生物化学与分子生物学报，2000，16(3)，382～387
9) Sachdev D, Chirgwin J M. Solubility of Proteins Isolated from Inclusion Bodies is Enhanced by Fusion to Maltose-binding Protein or Thioredoxin. Protein Expr Purif, 1998, 12:122～132
10) 徐明波，董小杰，孟文华等．原核基因工程中以包涵体形式表达产物的中试分离纯化研究．高技术通讯，1993，9：11～15
11) Babbin P C, West B L, Buechter D D et al. Removal of a Proteolytic Activity Associated with Aggregates Formed from Expression of Creatine Kinase in Escherichia Coli Leads to Improved Recovery of Active Enzyme. Biotechnology, 1990,8:945～949
12) 罗元明，牟颖，魏景艳．单链抗体2F3表达条件的优化及其提纯和性质研究．生物工程学报，2002，18(1)：74～79
13) 王进，华子春，方勇等．功率超声方法对蛋白质包涵体的解聚．生物化学与生物物理进展，1995，22(6)：543～545
14) 张治洲，张渝英．碱性复性条件下溶解包涵体可提高凝乳酶原复性率的机制．中国科学：C辑生命科学．1997，27(2)，103～108
15) 邹承鲁．第二遗传密码?-新生肽及蛋白质折叠的研究．湖南科学技术出
版社，1997，60～61
16) Maeda Y, Ueda T, Imoto T. Effective Renaturation of Denatured and Reduced Immunoglobulin G in vitro without Assistance of Chaperone. Protein Eng.,1996, 9, 95～100
17) De Bernardez Clark E. Refolding of Recombinant Proteins. Curr Opin Biotechnol, 1998, 9:157～163
18) Kuboi R, Morita S, Ota H et al. Protein Refolding Using Stimuli-responsive Polymer-modified Aqueous Two-phase Systems, J. Chromatogr. B, 2000, 743: 215-223
19) Batas B, Chaudhuri J B. Protein Refolding at High Concentration Using Size-exclusion Chromatography, Biotech. Bioeng., 1996, 50:16～23
20) Geng X D, Chang X Q, High-performance Hydrophobic Interaction Chromatography as a Tool for Protein Refolding. J. Chromatogr, 1992, 599: 185～194
21) Li M, Zhang G F, Su Z G, Dual Gradient Ion-Exchange Chromatography Improved Refolding Yield of Lysozyme. J. Chromatogr A, 2002, 959: 113～120
22) Zahn R, Human Prion Proteins Expressed in Escherichia Coli and Purified by High Affinity Column Refolding, FEBS Letters, 1997, 417, 3:400～404
23) Hagen A J, Hatton T A, Wang D I C. Protein Refolding in Reverse Micelles, Biotech. Bioeng., 1989, 35:955～965
24) Sachiko M, Setsuko O, Shi XH et al. Cycloamylose as an Efficient Artificial Chaperone for Protein Refolding. FEBS Letters, 2000, 486:131～135
25) Yoshimoto M, Shimanouchi T, Umakoshi H et al. Immobilized Liposome Chromatography for Refolding and Purification of Protein. J. Chromatogr. B, 2000, 743:93～99
26) 高永贵．溶菌酶和重组人干扰素-γ包涵体体外复性的研究．浙江大学博士学位论文．2002，p23～24
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