Renaturation Technology of Recombinant Fusion Protein Trx-IFN-CSP

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

Abstract

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

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.

References

[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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