福氏志贺痢疾杆菌硫胺素单磷酸激酶(ThiL)表达纯化及晶体生长研究  

Expression,Purification and Crystal Growth Studies on Thiamin Momophosphate Kinase (ThiL) from Shigella flexneri 2a Strain 301

作　　者：尚桂军[1] 丁志强[1] 赵子华[1] 聂荣鑫[2] 高伟[1] 仓怀兴[2] 

机构地区：[1]北京林业大学,北京100083 [2]中国科学院生物物理研究所,北京100101

出　　处：《中国生物工程杂志》2010年第12期25-29,共5页China Biotechnology

基　　金：国家"973"计划(07971002)资助项目

摘　　要：硫胺素单磷酸激酶(ThiL)在ATP存在下催化硫胺素单磷酸(TMP)形成硫胺素焦磷酸(TPP)和ADP,硫胺素焦磷酸就是维生素B1的活性形式。硫胺素单磷酸激酶属于一个小的ATP结合蛋白超家族成员。将来源于福氏志贺痢疾杆菌2a(301株)ThiL基因构建入pET-22b(+)表达载体,在大肠杆菌中得到高效表达,经过两步纯化,得到高纯度蛋白,用于晶体生长,经过对其晶体生长条件进行摸索和优化,得到了能用于X-射线衍射的单晶,为其结构解析、催化机理研究和药物设计提供了基础。Thiamin monophosphate kinase（ThiL）catalyzes the ATP-dependent phosphorylation of thiamin monophosphate（TMP）to form thiamin pyrophosphate（TPP）,the active form of vitamin B1.ThiL is a member of a small ATP binding superfamily.The gene of ThiL from Shigella flexneri 2a（strain 301）was constructed into the expression vector and over expressed in the E.coli.Then it was purified through two steps of chromatography leading to the high purity of the protein.The purified protein was screened for crystallization.The hit condition was optimized and gave rise to the single crystals for X-ray diffraction,which is the fundamental step for its structure determination,illumination of its catalytic mechanism,and corresponding drug design.

关 键 词：硫胺素单磷酸激酶(ThiL)晶体生长 福氏志贺痢疾杆菌2a301株 

分 类 号：Q786[生物学—分子生物学]