噬菌体phi29末端酶gp16蛋白C端结构域在溶液中的构象分析  

Solution Analysis of Phage phi29 gp16 Protein C terminal Domain

作　　者：武飞飞 蔡汝洁 陈婷[1] 张云龙[1] 陆昌瑞 WU Fei-Fei;CAI Ru-Jie;CHEN Ting;ZHANG Yun-Long;LU Chang-Rui(College of Chemistry,Chemical Engineering and Biotechnology,Donghua University,Shanghai 201620,China)

机构地区：[1]东华大学化学化工与生物工程学院,上海201620

出　　处：《生物化学与生物物理进展》2019年第3期305-312,共8页Progress In Biochemistry and Biophysics

基　　金：国家自然科学基金(31300603);中央高校基本科研基金(17D310512;2232014D3-39);励志计划(18D210501)资助项目~~

摘　　要：噬菌体phi29是一种双链DNA病毒,它的增殖和成熟过程需要将较大的子代DNA包装入一个空间极其有限的新生病毒衣壳中,这一步骤由其转运马达完成.转运马达具有三个部分:头颈连接器蛋白(the connector)、pRNA和ATP水解酶蛋白(gp16).在结构预测中发现gp16蛋白的C端结构域可能与pRNA或DNA结合,但尚未有相关文献可以证明这种相互作用,因此其结构的解析成为研究其功能的关键.本文利用大肠杆菌SUMO表达系统,对噬菌体phi29 gp16蛋白的C端结构域进行重组构建并诱导表达后,通过Ni-NTA亲和层析纯化,再用分子排阻色谱获得高纯度的目的蛋白质单体,纯度达到95%左右.最后用X射线小角散射技术(small angle X-ray scattering)对其构象进行分析,收集小角散射数据,当目的蛋白的浓度为1.3 g/L、所得到的目的蛋白的三维结构与同源蛋白FtsK的晶体结构高度拟合,且通过CRYSOL软件反推计算的曲线与实验曲线高度重合.通过原核表达、纯化及结构分析,成功获得了该结构域在溶液中的状态信息.这一研究不仅为后续关于该结构域的功能研究奠定基础,也为病毒感染的诊断和治疗提供新思路.Phage phi29, a double-stranded DNA virus, assembles its genome into its protein capsid to nearcrystalline density by a highly efficient molecular motor.The motor contains the connector, prohead RNA and ATP hydrolytic enzyme protein gpl6. Currently, no structure-function information is available regarding the C-terminal domain of gpl6. Our research aims to understand its role and interaction with pRNA or DNA. This study provides a method for recombinant gpl6 protein synthesis and purification by the E. coli SUMO expression system, and here we report the structural envelope of the C terminal domain of gpl6 obtained through small angle X-ray scattering (SAXS) technology. The gpl6 C temiinal domain gene was recombinantly expressed using the SUMO tag. The gpl6 gene was flanked by an N-terminal 6His-SUMO tag and purified by Ni-NTA affinity chromatography. High purity target protein monomer was obtained by size exclusion chromatography. This brings the purity of the target protein to about 95%. Then the concentration of the target protein to 1.3 g/L, and the SAXS data were collected at Shanghai Synchrotron Radiation Facility (SSRF) beamline BL19U. The resulting molecular envelop highly resembles the homologous protein FtsK, and the curves calculated by the CRYSOL software are highly coincident with the experimental curves. Through recombinant expression, purification and structure analysis, we obtained the solution structure of the gpl6. Structural infonnation derived from this study laid the foundation for future structural and functional research of gpl6 C teiminal, ultimately targeting the viral mechanisms for infection.

关 键 词：噬菌体phi29 gp16蛋白 C端结构域 X射线小角散射 

分 类 号：Q518.2[生物学—生物化学]