集成干扰素突变体IIFN72C的构建及其聚乙二醇定点修饰

中国生物工程杂志

2010, Vol. 30

Issue (04): 44-48

研究报告

集成干扰素突变体IIFN72C的构建及其聚乙二醇定点修饰

黄湘鹭¹,陶杉杉¹,徐晨^2**,李洁²,周建平¹

1.中国药科大学药学院南京 210009
2.北京三元基因工程有限公司北京 102600

Construction,Expression,Purification and Site-specific PEGylation of Integrated Interferon Mutant Cys72

HUANG Xiang-lu¹,TAO Shan-shan¹,XU Chen²,LI Jie²,ZHOU Jian-ping¹

1.Pharmaceutical Science Department，China Pharmaceutical University，Nanjing 210009，China
2.Beijing Tri-prime Genetic Engineering Co.,Ltd.，Beijing 102600，China

全文: PDF(452 KB) HTML

摘要：

目的：设计构建集成干扰素突变体IIFN72C并进行聚乙二醇定点修饰，以获得高活性的长效干扰素分子。方法：利用蛋白质分子同源模建，选择在集成干扰素分子IIFN的第72位引入半胱氨酸残基构成集成干扰素突变体IIFN72C。诱导表达后经包涵体变复性和层析纯化，与单甲氧基聚乙二醇（mPEGMAL）定点偶联。修饰产物经纯化后，以SDSPAGE考察其纯度，用WISHVSV系统进行生物活性测定。结果：IIFN72C以包涵体形式表达，表达量占菌体总蛋白的30%以上，比活性与突变前相当；修饰产物大多数为单修饰体，纯化后纯度大于98%，比活性保留约为修饰前的8%。结论：成功设计并表达IIFN72C用于PEG定点修饰，修饰产物活性保留得以提高。

关键词： 集成干扰素; 定点突变; 聚乙二醇定点修饰

Abstract:

Interferon alpha are used in clinic to treat a variety of viral diseases and cancers. They have short circulating half-life, which necessitates frequent administration to patients. Previous studies showed that it is possible to extend the circulating half-life of interferon alpha by modifying cysteine residues of the protein with poly(ethylene glycol)(PEG) reagents. But protein seldom have unpaired –SH, so a free cysteine residue was introduced into the protein by recombinant DNA technique and only 1% activity was retained after Pegylation. So a proper position should be selected for Pegylation and enhance the retained activity. Integrated Interferon (IIFN) has a wide variety of biological effects that include viral inhibition, antiproliferation, and immunomodulation . Aspartate residue at positon of 72 was chosed by homology sequence analysisi and space structure simulation methods. Integrated interferon mutant Cys72(IIFN72C) was a cysteine mutant of IIFN. The protein had higher specific activity and may used for site-directed modified by PEG. It was constructed by substitution of Asp at position 72 with Cys using site-directed mutagenesis. The DNA was constructed in pET-23b expression vector, and transformed into E.coli BL21(DE3). IIFN72C was expressed as inclusion bodies with yield of more than 30% of total bacterial protein. The recombinant protein was expressed by auto-induction and purified by DEAE Sepharose FF and Chelating Sepharose FF column chromatography. After purification, the protein was modified with a 20kDa mPEG-maleimide and the mono-PEGylated protein was separated from unmodified IIFN72C by stepwise elution. After purification, the purity of mono-PEG-IIFN72C was up to 98%, and the biological activity was more than 3×107IU/mg, The retained activity was up to 8%, which higher than before. These results confirmed the utility of site-specific PEGylation for creating long-acting interferon with higher activity.

Key words: Integrated interferon Site-directed mutagenesis Site-specific pegylation

收稿日期: 2010-02-10 出版日期: 2010-04-29

基金资助:

北京市科技计划重大专项（D0205001040321）资助项目

通讯作者: 徐晨 E-mail: xuchen@triprime.com

	服务
	把本文推荐给朋友
	加入引用管理器
	E-mail Alert
	RSS
	作者相关文章
	黄湘鹭
	陶杉杉
	徐晨
	李洁
	周建平

引用本文:

黄湘鹭陶杉杉徐晨李洁周建平. 集成干扰素突变体IIFN72C的构建及其聚乙二醇定点修饰[J]. 中国生物工程杂志, 2010, 30(04): 44-48.

HUANG Xiang-Lu, DAO Sha-Sha, XU Chen, LI Ji, ZHOU Jian-Beng. Construction,Expression,Purification and Site-specific PEGylation of Integrated Interferon Mutant Cys72. China Biotechnology, 2010, 30(04): 44-48.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2010/V30/I04/44

［1］刘耀波，杨轶，刘金毅,等. 新型α干扰素突变体及其制备方法.中国专利, CN1511849.20040714. Liu Y B，Yang Y，Liu J Y. New type of Interferonα mutant and preparing method. China，CN1511849.20040714.
［2］ Werner K，Bernard G，Alexander M，et al.The threedimensional high resolution structure of human interferon α2a determined by heteronuclear NMR spectroscopy in solution. Mol Biol，1997，274：661675.
［3］ Studier F W. Protein production by autoinduction in highdensity shaking cultures. Protein Expression and Purification，2005，41：207234.
［4］ Sims G E，Snape T J. A method for the estimation of polyethylene glycol in plasma protein fractions. Anal Biochem，1980，107：6063.
［5］姜忠义，高蓉，许松伟，等.药物蛋白的聚乙二醇修饰.中国药学杂志，2002,137(6):409412. Jiang Z Y, Gao R, Xu S W, et al. Chinese Pharmaceutical Journal，2002,137(6):409412.
［6］中国药典委员会编.《中华人民共和国药典》.北京：化学工业出版社,2005. Chinese pharmacopoeia commission.Pharmacopoeia of the Peoples Republic of China.Beijing:Chemical Industry Press,2005.
［7］ Glue P，Fang J W，RouzierPains R，et al. Pegylated interferon α2b：pharmacokinetics，pharmacodynamics，safety and preliminary efficacy data. Clin Pharmacol Ther，2000，68(5)：556567.
［8］ Ramon J，Saez V，Baez R，et al. PEGylated interferonalpha2b：abranched 40k polyethylene glycol derivative. Pham Res，2005，22(8) ：1374 1386.
［9］ Wang Y S，Youngster S，Grace M，et al. Structural and biological characterization of pegylated recombinant interferon alpha2b and its therapeutic implications. Advanced Drug Delivery Reviews， 2002，54：547570.
［10］ Foser S， Schacher A， Weyer K A， et al. Isolation， structural characterization， and antiviral activity of positional isomers of monopegylated interferon α2a (PEGASYS). Protein Expression and Purification，2003，30：78  87.
［11］ Zalipsky S，Lee C. Poly(ethylene glycol) Chemistry.in Biotechnical and Biomedical Applications. J M Harris ed, New York :Plenum Press, 1992. 347370.
［12］ Lee A C J，Powell J E，Tregear GW. A method for preparing βhCG COOH peptidecarrier conjugates of predictable composition. Mol Immunol，1980，17：747756.
［13］牛晓霞，周敏毅，刘金毅，等. 聚乙二醇定点修饰集成干扰素突变体II.中国生物工程杂志,2008,28(4):1720. Niu X X, Zhou M Y, Liu J Y, et al. China Biotechnology，2008,28(4) :1720.
［14］牛晓霞，刘金毅，杨轶，等.集成干扰素突变体Ⅱ的分子构建、表达及提纯.中国生物工程杂志,2006,26(12):15. Niu X X, Liu J Y, Yang Y, et al. China Biotechnology，2006,26(12):15.
［15］ Hu R，Bekisz J，Schmeisser H，et al. Human IFNα protein engineering： The amino acid residues at position 86 and 90 are important for antiproliferative activity. The Journal of Immunology，2001，167：14821489.
［16］ Chill J H，Nivasch R，Levy R， et al. The human interferon receptor：NMRbased modeling，mapping of the IFNalpha 2 binding site，and observed ligandinduced tightening.Biochemistry，2002，41(11)：35753585.
［17］ Uze G，Di Marco S，MouchelVielh E，et al. Domains of interaction between alpha interferon and its receptor components. Mol Biol，1994，243(2)：245257.

[1]	郭芳,张良,冯旭东,李春. 植物源UDP-糖基转移酶及其分子改造^*[J]. 中国生物工程杂志, 2021, 41(9): 78-91.
[2]	彭向雷,王烨,王丽男,苏彦斌,付远辉,郑妍鹏,何金生. 单引物PCR法引入定点突变 ^*[J]. 中国生物工程杂志, 2020, 40(8): 19-23.
[3]	赵晓艳,陈允妲,章雅倩,吴晓玉,王飞,陈金印. **Myxococcus sp.V11海藻糖合酶TreS II分子改造 ^***[J]. 中国生物工程杂志, 2020, 40(3): 79-87.
[4]	苏永君,胡蝶,胡博淳,李闯,文正,章晨,邬敏辰. 定点突变提高环氧化物水解酶AuEH2催化对甲基苯基缩水甘油醚的对映选择性^*[J]. 中国生物工程杂志, 2020, 40(3): 88-95.
[5]	阚婷婷,宗迅成,苏永君,王婷婷,李闯,胡蝶,邬敏辰. 定点突变改善PvEH1对邻甲基苯基缩水甘油醚的催化特性 ^*[J]. 中国生物工程杂志, 2019, 39(6): 9-16.
[6]	孟浩毅,李丹阳,孙正阳,杨兆勇,张志斐,袁丽杰. 人类线粒体肌酸激酶uMtCK的底物结合位点分析 ^*[J]. 中国生物工程杂志, 2018, 38(5): 24-32.
[7]	李雪晴, 袁风娇, 程建青, 董运海, 李剑芳, 邬敏辰. 杂合β-甘露聚糖酶AuMan5A^loop的H321对其酶学性质的影响[J]. 中国生物工程杂志, 2017, 37(2): 48-53.
[8]	吴芹, 胡蝶, 李雪晴, 袁风娇, 李剑芳, 邬敏辰. Y13F定点突变改良米曲霉中温木聚糖酶的耐热性[J]. 中国生物工程杂志, 2016, 36(12): 36-41.
[9]	代玉环, 徐尧, 罗颖, 代洋, 石伟林, 徐瑶. Myocardin调控心肌H9C2细胞Ca²⁺通道机制研究[J]. 中国生物工程杂志, 2016, 36(11): 1-6.
[10]	向缅, 朱建全, 俞继华, 李洋洋, 李娟娟, 刘祖碧, 王万军, 廖海, 周嘉裕. 决明胰蛋白酶抑制剂1活性相关残基的定点突变与抑制活性分析[J]. 中国生物工程杂志, 2016, 36(10): 15-20.
[11]	李瑶瑶, 毕静, 王艺红, 秦云贺, 张雪莲. 乙酰化修饰调控结核杆菌异柠檬酸裂合酶的研究[J]. 中国生物工程杂志, 2015, 35(6): 8-13.
[12]	高瑞平, 程隆斌, 李振秋. 一种简便快速的单引物PCR定点突变方法[J]. 中国生物工程杂志, 2015, 35(5): 61-65.
[13]	夏亚穆, 李晨晨. 环糊精葡萄糖基转移酶的基因改造与高效表达[J]. 中国生物工程杂志, 2015, 35(2): 105-110.
[14]	阮景军, 杨毅, 唐自钟, 陈惠. 基于定点突变技术对苦荞麦胰蛋白酶抑制剂活性位点的研究[J]. 中国生物工程杂志, 2015, 35(12): 30-36.
[15]	裴智勇, 侯仙慧, 桂小柯, 陈禹保. Primer Spanner:一个高效的定点突变PCR引物设计在线工具[J]. 中国生物工程杂志, 2015, 35(10): 53-58.

Viewed

Full text

Abstract

Cited

Shared

Discussed