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化工学报  2015 

重组瑞替普酶包涵体制备及其体外复性

DOI: 10.11949/j.issn.0438-1157.20141141, PP. 709-716

Keywords: 生物分离,蛋白质复性,二硫键,正交实验设计,瑞替普酶

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Abstract:

将携带重组瑞替普酶(reteplase,rt-PA)的质粒成功转化到大肠杆菌BL21(DE3)后,诱导表达获得包涵体,考察了诱导剂浓度、培养温度和培养时间等条件对目标蛋白表达量的影响。在此基础上,对高效表达的rt-PA包涵体体外复性过程进行了详细研究。首先利用单因素实验考察了复性液pH、GSH浓度、GSH/GSSG比例、蛋白浓度等各种复性条件对复性效果的影响;并结合正交实验设计,进一步研究了高蛋白浓度下复性后rt-PA酶活变化情况。以0.2mmol·L-1IPTG诱导,在33℃下培养6h,每升发酵液约可获得1.7g粗制包涵体。适宜的复性条件为蛋白浓度50mg·ml-1,pH10.0,GSH浓度1mmol·L-1,GSH/GSSG比例8,复性收率为87.2%。影响高蛋白浓度下rt-PA复性的关键因素为复性液初始pH及GSH浓度,在800mg·ml-1蛋白浓度下复性后rt-PA比活可达7.54×104IU·mg-1,荧光光谱分析结果表明复性后rt-PA恢复了其天然态结构。

References

[1]  Nicholas J A. Management of postoperative complications: cardiovascular disease and volume management [J]. Clinics in Geriatric Medicine, 2014, 30(2): 293-301
[2]  Gawryszewski V P, de Souza M. Mortality due to cardiovascular diseases in the Americas by region, 2000—2009 [J]. Sao Paulo Medical Journal, 2014, 132(2): 105-110
[3]  Pennica D, Holmes W E, Kohr W J, Harkins R N, Vehar G A, Ward C A, Bennett W F, Yelverton E, Seeburg P H, Heyneker H L, Goeddel D V, Collen D. Cloning and expression of human tissue-type plasminogen activator cDNA in E. coli [J]. Nature, 1983, 301(5897): 214-221
[4]  Kohnert U, Rudolph R, Verheijen J H, Weening-Verhoeff E J, Stern A, Opitz U, Martin U, Lill H, Prinz H, Lechner M, Kresse G B, Buckel P, Fischer S. Biochemical-properties of the kringle 2 and protease domains are maintained in the refolded t-PA deletion variant BM 06.022 [J]. Protein Engineering, 1992, 5(1): 93-100
[5]  Topol E, Califf R, Ohman E. A comparison of reteplase with alteplase for acute myocardial infarction [J]. New England Journal of Medicine, 1997, 337(16): 1118-1123
[6]  Gao L, Zhang C, Li L L, Liang L, Deng X, Wu W T, Su Z G, Yu R. Construction, expression and refolding of a bifunctional fusion protein consisting of C-terminal 12-residue of hirudin-PA and reteplase [J]. Protein Journal, 2012, 31(4): 328-336
[7]  Obukowicz M G, Gustafson M E, Junger K D, Leimgruber R M, Wittwer A J. Secretion of active kringle-2-serine protease in Escherichia coli [J]. Biochemistry, 1990, 29(41): 9737-9745
[8]  Harris T, Patel T, Marston F, Little S, Emtage J S, Opdenakker G, Volckaert G, Rombauts W, Billiau A, Desomer P. Cloning of cDNA coding for human tissue-type plasminogen-activator and its expression in Escherichia coli [J]. Molecular Biology & Medicine, 1986, 3(3): 279-292
[9]  Feng Changgen(冯长根), Chen Man(陈嫚),Ren Qisheng(任启生),Song Xinrong(宋新荣). Study on expression, renaturation and purification of reteplase [J]. Chin. Pharm. J. (中国药学杂志), 2004(7): 74-76
[10]  Khodabakhsh F, Zia M F, Moazen F, Rabbani M, Sadeghi H. Comparison of the cytoplasmic and periplasmic production of reteplase in Escherichia coli [J]. Preparative Biochemistry & Biotechnology, 2013, 43(7): 613-623
[11]  Sambrook J, Russell D W. Molecular Cloning - A Laboratory Manual [M]. 3rd ed. New York: Cold Spring Harbor Laboratory Press, 2001
[12]  Bradford M M. Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein-dye binding [J]. Analytical Biochemistry, 1976, 72(1/2): 248-254
[13]  Luo X G, Tian W J, Ni M, Jing X L, Lv L H, Wang N, Jiang Y, Zhang T C. Soluble expression of active recombinant human tissue plasminogen activator derivative (K2S) in Escherichia coli [J]. Pharmaceutical Biology, 2011, 49(6): 653-657
[14]  Sun Haibo(孙海波), Liang Bufeng(梁布峰), Wang Junyan(王君艳),Ling Jianya(凌建亚), Wang Ge(王革). Comparative study on assay methods for reteplase activity [J]. Food and Drug (食品与药品), 2007(9): 4-7
[15]  Yu Rong(余蓉), Zhang Guifeng(张贵锋), Gao Ling(高玲), Su Zhiguo(苏志国), Wu Wutong(吴梧桐). Primary structure determination of hirudin and reteplase fusion protein by LC/ESI-MS/MS spectrometry [J]. Acta Pharmaceutica Sinica(药学学报), 2008(7): 737-742
[16]  Fischer B, Sumner I, Goodenough P. Isolation, renaturation and formation of disulfide bonds of eukaryotic proteins expressed in Escherichia coli as inclusion-bodies [J]. Biotechnology & Bioengineering, 1993, 41(1): 3-13
[17]  Guise A D, West S M, Chaudhuri J B. Protein folding in vivo and renaturation of recombinant proteins from inclusion bodies [J]. Molecular Biotechnology, 1996, 6(1): 53-64
[18]  Lin X L, Umetsu T. The high pH and pH-shift refolding technology [J]. Current Pharmaceutical Biotechnology, 2010, 11(3): 293-299
[19]  Luo M, Guan Y X, Yao S J. Statistical optimization and multiple objective programming of lysozyme refolding catalyzed by recombinant DsbA in vitro [J]. Process Biochemistry, 2012, 47(8): 1268-1276
[20]  Huang Shoukun(黄寿锟), Guan Yixin(关怡新), Yu Hongwei(于洪巍), Yao Shanjing(姚善泾). Refolding of recombinant pig liver esterase inclusion bodies in vitro assisted by thiol-carrying latex particles [J]. CIESC Journal(化工学报), 2014, 65(1): 305-312
[21]  Guan Yixin(关怡新), Fei Zhengzheng(费峥峥), Luo Man(罗曼), Yao Shanjing(姚善泾). Minichaperone(GroEL191~345)-mediated in vitro refolding of recombinant human interferon-γ inclusion body [J]. Prog. Biochem. Biophys.(生物化学与生物物理进展), 2004(10): 907-911
[22]  Chung F, Salehi J A, Wei V K. Optical orthogonal codes - design, analysis and applications [J]. IEEE Transactions on Information Theory, 1989, 35(3): 595-604
[23]  Li Lin(李琳), Dong Xiaoyan(董晓燕), Sun Yan(孙彦). Dynamic fed-batch refolding behavior of denatured-reduced lysozyme at high concentration [J]. Journal of Chemical Industry and Engineering (China)(化工学报), 2003, 54(12): 1719-1723

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