来源于Mangifera indica L.C-糖基转移酶MiCGTb的大肠杆菌原核表达、纯化、结晶化以及晶体学研究  

Prokaryotic expression,purification and crystallization of C-glycosyltransferases from Mangifera indica L.and the preliminary crystallographic study

作　　者：樊帅[1] 吕广新 刘凡[1] 金媛媛[1] 杨兆勇[1] FAN Shuai;LYU Guang-xin;LIU Fan;JIN Yuan-yuan;YANG Zhao-yong(Institute of Medicinal Biotechnology,Chinese Academy of Medical Sciences&Peking Union Medical College,Beijing 100050,China)

机构地区：[1]中国医学科学院北京协和医学院医药生物技术研究所微生物代谢工程研究室,北京100050

出　　处：《中国医药生物技术》2021年第5期385-390,共6页Chinese Medicinal Biotechnology

基　　金：中国医学科学院医学与健康科技创新工程(2019-I2M-1-005);国家自然科学基金面上项目(81872782)。

摘　　要：目的获得来源于Mangifera indica L.C-糖基转移酶C-glycosyltransferase(MiCGTb)的晶体结构,为解析C-糖基转移酶催化机制提供结构基础。方法因MiCGTb较不稳定,构建MiCGTb截短蛋白MiCGTb1(Asn8-Lys465)表达质粒,利用大肠杆菌表达系统实现MiCGTb1可溶性表达,采用亲和层析、离子交换层析和分子排阻色谱纯化目的蛋白,选用气相扩散悬滴法,通过优化结晶条件获得可进行X-ray衍射的高质量晶体,获取衍射数据。结果运用分子生物学方法成功构建含MiCGTb1基因的重组质粒,转化BL21-CodonPlus(DE3)-RIPL得到重组表达菌株。运用亲和层析和离子交换层析蛋白纯化技术,获得用于结晶化的蛋白样品。气相扩散悬滴法确定的结晶条件为:0.2 mol/L MgCl_(2)、0.1 mol/L Tris pH 8.0、40%MPD,结晶温度为23℃,添加剂为乙二醇(0.75%v/v),获得X-ray衍射分辨率为3.30Å的衍射数据。结论来源于Mangifera indica L.C-糖基转移酶截短蛋白MiCGTb1晶体的获得有望对解析C-糖基转移酶的晶体结构以及催化机制奠定基础。Objective We aim to solve the crystal structures of MiCGTb from Mangifera indica L.to understand the structural basis for the mechanism of C-glycosylation.Methods Since MiCGTb is unstable,the truncated MiCGTb1(Asn8-Lys465)was constructed in this study.The soluble expression of MiCGTb1 protein was obtained by Escherichia coli expression system.The target protein was purified by affinity chromatography and ion exchange chromatography in order to obtain MiCGTb1.High quality crystal for X-ray diffraction was obtained by optimizing crystallization conditions by employing vapor diffusion suspension method.Results The recombinant plasmid containing MiCGTb1 gene was successfully constructed by molecular biological method,and the strain BL21-CodonPlus(DE3)-RIPL with high expression of MiCGTb1 protein was obtained by optimization.After the purification of the MiCGTb1 through affinity chromatography and ion exchange chromatography,it was used for crystallization and structure determination.The crystal of MiCGTb1 was obtained at optimum conditions(0.2 mol/L MgCl_(2),0.1 mol/L Tris pH 8.0,40%MPD,0.75%v/v ethandial,23℃),and the resolution of diffraction data up to 3.30Å.Conclusion The obtained crystal structure of MiCGTb1 is expected to provide guidance and basis for the elucidating catalytic mechanism of C-glycosyltransferases.

关 键 词：C-糖基转移酶 蛋白纯化 结晶 X-射线衍射 

分 类 号：R37[医药卫生—病原生物学]