重组融合蛋白Trx-IFN-CSP复性工艺研究

doi:10.13560/j.cnki.biotech.bull.1985.2016.06.032

生物技术通报 ›› 2016, Vol. 32 ›› Issue (6): 219-225.doi: 10.13560/j.cnki.biotech.bull.1985.2016.06.032

重组融合蛋白Trx-IFN-CSP复性工艺研究

黄演婷^1,2,3, 卢雪梅^2,3, 杨小蓉¹, 金小宝^2,3, 朱家勇^2,3

1. 广东药学院附属第一医院，广州 510006
2. 广东药学院基础学院药用生物活性物质研究所，广州 510006
3. 广东省生物活性药物研究重点实验室，广州 510006

收稿日期:2015-08-02 出版日期:2016-06-27 发布日期:2016-06-28
作者简介:黄演婷，女，硕士,研究方向：药用生物活性物质结构、功能与应用；E-mail：olive1987@aliyun.com
基金资助:
国家科技部新药创制重大专项项目(2013ZX09103003-003)

Renaturation Technology of Recombinant Fusion Protein Trx-IFN-CSP

HUANG Yan-ting^1，2，3, LU Xue-mei^2，3, YANG Xiao-rong¹, JIN Xiao-bao^2，3, ZHU Jia-yong^2,3

1. The First Affiliated Hospital of Guangdong Pharmaceutical University，Guangzhou 510006
2. Institution of Pharmaceutical Bioactive Substances，School of Basic Courses，Guangdong Pharmaceutical University，Guangzhou 510006
3.Guangdong Key Laboratory of Pharmaceutical Bioactive Substances，Guangzhou 510006

Received:2015-08-02 Published:2016-06-27 Online:2016-06-28

摘要/Abstract

摘要： 旨在建立并优化融合蛋白Trx-IFN-CSP的复性工艺。对重组融合Trx-IFN-CSP进行体外复性研究，考查pH、温度、蛋白浓度、氧化还原体系及辅助复性小分子等复性条件对融合蛋白重折叠的影响。结果显示，适合Trx-IFN-CSP复性的方法为，反复冻融联合超声破菌获得包涵体；用含有1% TritonX-100、2 mol/L尿素、2% DOC洗涤液初步纯化包涵体；再用6 mol/L盐酸胍溶解液变性包涵体；脉冲加样稀释变性液后4℃条件下梯度透析复性，使用L-Arg辅助复性。经肠激酶切去Trx标签后，每升发酵液最终获得110-130 mg肝靶向干扰素，每批蛋白纯度都在95%以上，比活性在1.9-2.4×10⁸ U/mg之间，制备工艺稳定。

关键词: 融合蛋白, 肝靶向干扰素, 包涵体, 脉冲稀释, 梯度透析

Abstract: This work is to establish and optimize the method of refolding fusion protein Trx-IFN-CSP. The refolding of recombinant protein in vitro was studied，i.e.，investigating the effects of pH，temperature，protein concentration，redox systems and auxiliary refolding molecules on the refolding process of fusion protein. The results were as below. The proper measure to refold the Trx-IFN-CSP was to obtain the inclusion bodies by repeated combined freeze-thaw with ultrasonic to crack bacteria. The inclusion bodies were preliminarily purified using scrub solution of 1％ TritonX-100，2 mol/L urea，and 2% DOC，and then denatured in 6 mol/L guanidine hydrochloride. After the pulse dilution，the renaturation was conducted under 4℃ by gradient dialysis with the assistance of L-Arg. After the Trx-tag was removed by recombinant enterokinase digestion，approximately 110-130 mg of the pure recombinant liver-targeted interferon was obtained from 1 L Escherichia coli culture. Each batch of protein had a purity of over 95% and antibacterial activities were about 1.9-2.4×10⁸ U/mg，thus the technology of preparation was stable.

Key words: fusion protein, liver-targeted interferon, inclusion body, pulse dilution, gradient dialysis

黄演婷, 卢雪梅, 杨小蓉, 金小宝, 朱家勇. 重组融合蛋白Trx-IFN-CSP复性工艺研究[J]. 生物技术通报, 2016, 32(6): 219-225.

HUANG Yan-ting, LU Xue-mei, YANG Xiao-rong, JIN Xiao-bao, ZHU Jia-yong. Renaturation Technology of Recombinant Fusion Protein Trx-IFN-CSP[J]. Biotechnology Bulletin, 2016, 32(6): 219-225.

参考文献

[1]Mayer M, Buchner J. Refolding of inclusion body proteins[J]. Methods Mol Med, 2004, 94(1)：239-254.
[2]Anfinsen CB. Principles that govern the folding of protein chains[J]. Science, 1973, 181(4096)：223-230.
[3]Jungbauer A, Kaar W. Current status of technical protein refolding[J]. Journal of Biotechnology, 2007, 128(3)：587-596.
[4] De Bemardez, Clark E. Protein refolding for industrial processes[J]. Current Opinion in Biotechnology, 2001, 12(2)：202-207.
[5]Katoh S, Sezai Y, Yamaguchi T, et al. Refolding of enzymes in a fed-batch operation[J]. Process Biochem, 1999, 35：297-300.
[6]李明. 离子交换色谱复性蛋白质[D]. 北京：中国科学院2003.
[7]Maeda Y, Ueda T, Yamada H, et al. The role of net chagre on the renautration of reduced lysozyme by the sulfhydryl-disulfide interchange reaction[J]. Protein Engineering, 1994, 7(10)：1249-1254.
[8]Guise AD, West S lI, Chaudhuri AB. Protein folding in Vivo and renaturation of recombinant proteins from inclusion bodies[J]. Mol Biotechnol, 1996, 6(1)：53-64.
[9]Kiefhaber T. Protein aggregation in vitro and in vivo. aquantitive model of the kinetic competition between folding and aggregation[J]. Biotechnology, 1991, 9(9)：825-829.
[10] Wetlaufer DB, Xie Y. Control of aggregation in protein refolding：a variety of surfactants promote renaturation of carbonic anhydrase II[J]. Protein Sci, 1995, 4(8)：1535-1543.
[11]Hamada H, Shiraki K. L-argininamide improves the refolding more effectively than L-arginine[J]. J Biotechnol, 2007, 130(2)：153-160.
[12]Fischer S, Rudolph R, Mattes R. Process for the activation of genetechnologically produced heterologous eukayrotic proteins after expression in prokayrotes[J]. Europen Patent, 1986, 393：725AI
[13]Buchner J, Pastan I, Brinkmann U. A method for increasing the yield of properly folded recombinant fusion proteins：single-chain immunotoxins from renuatration of bacterial inclusion bodies[J]. Anal Biochem, 1992, 205(2)：263-270.

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重组融合蛋白Trx-IFN-CSP复性工艺研究

Renaturation Technology of Recombinant Fusion Protein Trx-IFN-CSP

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