<em>E.coli</em> T蛋白CM的原核表达及核磁方法指导下的晶体优化

中国生物工程杂志

2013, Vol. 33

Issue (9): 38-44

技术与方法

E.coli T蛋白CM的原核表达及核磁方法指导下的晶体优化

芮斌, 谢长林, 江娜, 文汉

安徽农业大学生命科学学院合肥 230036

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

RUI Bin, XIE Chang-lin, JIANG Na, WEN Han

School of Life Sciences, Anhui Agricultural University, Hefei 230036, China

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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。

关键词： 分支酸变位酶; CM_T; 原核表达; HSQC; 晶体优化

Abstract: 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 CM_T was expressed in competent cells E.coli BL21-Gold(DE3) successfully. The purity could reach electrophoresis purity after purification by Ni-NTA affinity chromatography, GE superdex75 gel filtration column and GE MonoQ cation-exchange chromatography. The result of mass spectra matched with the original sequence of CM_Tbasically. And then CM_T crystal were found in the condition of 30% PEG 4000,100mmol/L NaAc pH4.6,200mmol/L NH₄Ac. The application of ¹H-¹⁵N HSQC NMR provided guidance for optimization of CM_Tcrystal, and finally we successfully obtained the high diffraction CM_T crystal. This work lays the foundation for the structure of CM_T.

Key words: CM CM_T Prokaryotic expression HSQC Crystal optimization

收稿日期: 2013-04-17 出版日期: 2013-09-25

ZTFLH:

TQ138

通讯作者: 文汉lifwen1000@163.com E-mail: lifwen1000@163.com

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引用本文:

芮斌, 谢长林, 江娜, 文汉. E.coli T蛋白CM的原核表达及核磁方法指导下的晶体优化[J]. 中国生物工程杂志, 2013, 33(9): 38-44.

RUI Bin, XIE Chang-lin, JIANG Na, WEN Han. Prokaryotic Expression of CM of T-protein from E.coli and Its Crystal Optimization Guided by NMR. China Biotechnology, 2013, 33(9): 38-44.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2013/V33/I9/38

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