E.coliT蛋白CM的原核表达及核磁方法指导下的晶体优化  

Prokaryotic Expression of CM of T-protein from E. coli and Its Crystal Optimization Guided by NMR

作　　者：芮斌[1] 谢长林[1] 江娜[1] 文汉[1] 

机构地区：[1]安徽农业大学生命科学学院,合肥230036

出　　处：《中国生物工程杂志》2013年第9期38-44,共7页China Biotechnology

摘　　要：越来越多的研究表明许多致病菌的病原性与生物体内莽草酸途径中的分支酸变位酶(chorismate mutase,CM)有着密切的关系,因此CM成为了开发新型抗生素和抗菌药物的热门靶标酶。采用分子克隆的方法,在感受态细胞BL21-Gold(DE3)中重组表达大肠杆菌(E.coli)T蛋白的CM(CM T)。通过Ni-NTA亲和层析柱、GE Superdex75凝胶过滤层析柱和GE MonoQ阳离子交换层析柱纯化后得到电泳纯的重组蛋白,质谱鉴定结果与原始序列基本吻合。经过蛋白质晶体试剂盒的初步筛选,在30%PEG 4000,100mmol/L NaAc pH4.6,200mmol/L NH4Ac条件中发现CM T晶体。利用核磁共振方法,分析CM T的1H-15N HSQC谱图信息,指导设计CM T晶体的优化方案,成功获得高衍射能力的CM T晶体,为CM T结构的解析奠定了坚实的基础。More and more researches have shown that the pathogenicities of many bacteria are related to their chorismate mutases in the shikimate pathway. CMs represent a hot selective target for the development of antibiotics and fungicides. By PCR the recombinant CMT was expressed in competent cells E. coli BL21-Gold （ DE3 ） successfully. chromatography, GE result of mass spectra the condition of 30% HSQC NMR provided The purity could reach electrophoresis purity after purification by Ni-NTA affinity superdex75 gel filtration column and GE MonoQ cation-exchange chromatography. The matched with the original sequence of CMTbasically. And then CMT crystal were found in PEG 4000, 100mmoL/L NaAc pH4.6,200mmol/L NH4Ac. The application of IH-15N guidance for optimization of CMT crystal, and finally we successfully obtained the high diffraction CMT crystal. This work lays the foundation for the structure of CMT

关 键 词：分支酸变位酶 CMT 原核表达 HSQC 晶体优化 

分 类 号：TQ138[化学工程—无机化工]