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  

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

作　　者：张宸 陈韶华[2] 吴文倩[2] 周建芹 ZHANG Chen CHEN Shao-hua WU Wen-qian ZHOU Jian-qin(College of Pharmaceutical Sciences, Soochow University, Suzhou 215123, China Experimental Center of Medical College, Soochow University, Suzhou 215123, China)

机构地区：[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

出　　处：《中国生物工程杂志》2017年第9期82-88,共7页China Biotechnology

摘　　要：研究微生物谷氨酰胺转氨酶(mTG)催化细胞色素c(Cytc)的PEG定点修饰的可行性,并优化修饰条件,研究PEG修饰对Cytc性质的影响。将单甲氧基聚乙二醇氨(mPEG-NH_2)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-NH_2、CBZQG-mPEG(mTG)修饰Cytc,研究酶法定点修饰Cytc残基的可行性;改变酶的用量、温度、反应时间和p H等反应条件优化谷胺酰胺转氨酶催化修饰Cytc的条件。研究结果表明:(1)mPEG-NH_2不能作为mTG的底物修饰Cytc,甲氧基聚乙二醇氨(mPEG-NH_2)分子上引入谷氨酰胺残基后,在mTG的催化作用下了实现Cytc的PEG修饰,而且基于mTG的底物特异性实现了PEG定点修饰Cytc的赖氨酸(Lys)残基;(2)37℃温度下,p H 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Cytc产物复杂,是多种多点修饰产物的混合物,酶法催化PEG修饰Cytc只产生单一产物;(4)与天然Cytc相比,修饰后Cytc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。Experiments were carried out to investigate the possibilities of site specific PEGylation of therapeutic proteins（ cytochrome c,Cytc） catalyzed by mTG. Then reaction conditions were optimized and the properties of the PEGylated Cytc were explored. CBZ-QG-mPEG was successfully synthesized by introducing CBZQG 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 Cytc catalyzed by mTG were investigated. CBZ-QG-mPEG was coupled to the specific Lys residue of Cytc catalyzed by mTG. The optimized PEGylationconditions were as follows： 37℃,pH 8. 0,mTG 1. 0mg/ml,reaction time 2h. The PEGylated Cytc exhibited better thermal stability and pH stability than the native Cytc. Only one specific Lys residue of Cytc was PEGylated catalyzed by mTG,while several Lys residues of Cytc were conjugated with mPEG-SPA by using the chemical method,which leading to the heterogeneity of the derivatives. The prominent advantage of mTGmediated 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.

关 键 词：谷胺酰胺转氨酶 细胞色素C 定点修饰 

分 类 号：Q814.9[生物学—生物工程]