微生物谷氨酰胺转氨酶催化细胞色素c赖氨酸残基的定点修饰

doi:10.13523/j.cb.20170911

中国生物工程杂志

2017, Vol. 37

Issue (9): 82-88 DOI: 10.13523/j.cb.20170911

技术与方法

微生物谷氨酰胺转氨酶催化细胞色素c赖氨酸残基的定点修饰

张宸¹, 陈韶华², 吴文倩², 周建芹¹

1. 苏州大学医学部药学院苏州 215123;
2. 苏州大学医学部实验中心苏州 215123

Modifiation of Cytochrome c at the Level of Lysine Residues Mediated by Microbial Transglutaminase

ZHANG Chen¹, CHEN Shao-hua², WU Wen-qian², ZHOU Jian-qin¹

1. College of Pharmaceutical Sciences, Soochow University, Suzhou 215123, China;
2. Experimental Center of Medical College, Soochow University, Suzhou 215123, China

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摘要： 研究微生物谷氨酰胺转氨酶（mTG）催化细胞色素c（Cyt c）的PEG定点修饰的可行性，并优化修饰条件，研究PEG修饰对Cyt c性质的影响。将单甲氧基聚乙二醇氨（mPEG-NH₂）与N-苄氧羰基-谷氨酰胺-甘氨酸（CBZ-QG）共价结合制备含谷氨酰胺残基的甲氧基聚乙二醇衍生物（N-苄氧羰基-谷氨酰胺-甘氨酰-单甲氧基聚乙二醇，CBZ-QG-mPEG）；mTG分别催化mPEG-NH₂、CBZ-QG-mPEG（mTG）修饰Cyt c，研究酶法定点修饰Cyt c残基的可行性；改变酶的用量、温度、反应时间和pH等反应条件优化谷胺酰胺转氨酶催化修饰Cyt c的条件。研究结果表明：①mPEG-NH₂不能作为mTG的底物修饰Cyt c，甲氧基聚乙二醇氨（mPEG-NH₂）分子上引入谷氨酰胺残基后，在mTG的催化作用下了实现Cyt c的PEG修饰，而且基于mTG的底物特异性实现了PEG定点修饰Cyt c的赖氨酸（Lys）残基；②37℃温度下，pH 8.0的溶液中，1mg/ml的mTG催化修饰反应2h是最佳修饰反应条件；③化学法PEG修饰Cyt c产物复杂，是多种多点修饰产物的混合物，酶法催化PEG修饰Cyt c只产生单一产物；④与天然Cyt c相比，修饰后Cyt c的活力、稳定性都有所提高。提出的谷胺酰胺转胺酶催化修饰法解决了蛋白质Lys残基难以定点修饰的难题，拓展了mTG在蛋白质修饰方面的应用。

关键词： 定点修饰; 谷胺酰胺转氨酶; 细胞色素c

Abstract: Experiments were carried out to investigate the possibilities of site specific PEGylation of therapeutic proteins (cytochrome c, Cyt c) catalyzed by mTG.Then reaction conditions were optimized and the properties of the PEGylated Cyt c were explored. CBZ-QG-mPEG was successfully synthesized by introducing CBZ-QG into methoxypolyethylene glycol amine (mPEG-NH₂) and could act as the acyl donor of mTG.The possibilities of mPEG-NH₂ acting as an amino donor or CBZ-QG-mPEGacting as an acyl donor to modify Cyt c catalyzed by mTG were investigated. CBZ-QG-mPEG was coupled to the specific Lys residue of Cyt c catalyzed by mTG.The optimized PEGylationconditions were as follows:37℃, pH 8.0, mTG 1.0mg/ml, reaction time 2h. The PEGylated Cyt c exhibited better thermal stability and pH stability than the native Cyt c. Only one specific Lys residue of Cyt c was PEGylated catalyzed by mTG, while several Lys residues of Cyt c were conjugated with mPEG-SPA by using the chemical method, which leading to the heterogeneity of the derivatives. The prominent advantage of mTG-mediated catalysis is its highsite specificity and thus homogeneity.Here, the mTG-mediated PEGylation of proteins at the level of lysine (Lys) residues was developed, which overcome the random modification of Lys residues by the chemical method andwas expected to be generally applied to protein modification.

Key words: Site specific modification Cytochrome c Transglutaminase

收稿日期: 2017-02-14 出版日期: 2017-09-25

ZTFLH:

Q814.9

通讯作者: 周建芹 E-mail: zhoujianqin@suda.edu.cn

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引用本文:

张宸, 陈韶华, 吴文倩, 周建芹. 微生物谷氨酰胺转氨酶催化细胞色素c赖氨酸残基的定点修饰[J]. 中国生物工程杂志, 2017, 37(9): 82-88.

ZHANG Chen, CHEN Shao-hua, WU Wen-qian, ZHOU Jian-qin. Modifiation of Cytochrome c at the Level of Lysine Residues Mediated by Microbial Transglutaminase. China Biotechnology, 2017, 37(9): 82-88.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20170911 或 https://manu60.magtech.com.cn/biotech/CN/Y2017/V37/I9/82

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