人ZRANB3蛋白在大肠埃希菌中的表达纯化和功能研究  

Expression,purification and function of human ZRANB3 protein in Escherichia coli

作　　者：郑琳 向嵩 陈朴 ZHENG Lin;XIANG Song;CHEN Pu(Department of Biochemistry and Molecular Biology,School of Basic Medicine,Tianjin Medical University,Tianjin 300070,China)

机构地区：[1]天津医科大学基础医学院生物化学与分子生物学系,天津300070

出　　处：《中国病原生物学杂志》2024年第9期1031-1035,1041,共6页Journal of Pathogen Biology

基　　金：天津市教委科研计划项目(自然科学)(No.2021KJ249)。

摘　　要：目的人体细胞中,ZRANB3蛋白在DNA损伤应对通路中发挥重要功能。目前,对ZRANB3的结构及其发挥功能的机制还了解甚少。为开展对ZRANB3蛋白结构和功能的研究,急需一种大规模制备高纯度、折叠正确ZRANB3的方法。ZRANB3是大分子量、多结构域的人源蛋白,这类蛋白的表达和纯化一般较为困难。本研究的目的是建立一种高效的ZRANB3的表达和纯化方法。方法通过基因克隆技术将经过密码子优化的ZRANB3基因插入到pET.28a载体中,构建重组质粒;将重组质粒转化至大肠埃希菌Rosetta菌株中,用IPTG诱导ZRANB3表达;采用亲和层析、离子交换层析和凝胶过滤层析等多种层析手段纯化ZRANB3并通过SDS-PAGE鉴定目的蛋白的纯度。通过核酸内切酶实验和复制叉退行实验对重组蛋白ZRANB3的功能进行验证。结果本研究建立了从大肠埃希菌中制备人源ZRANB3的方案,有效地纯化了ZRANB3蛋白,获得了较大量高纯度的ZRANB3蛋白,并且通过体外功能实验证明了制备的人源重组蛋白ZRANB3具有被报导的生化活性,包括ATP依赖的核酸内切酶活性和催化复制叉退行的活性。通过本研究建立的方法,可从9L的大肠埃希菌培养液中制备约0.5 mg纯度大于95%的、具有生化活性的ZRANB3蛋白。结论本研究提供了一种高效制备大量、高纯度且具有酶活性的人源ZRANB3蛋白的方法,为深入理解其促进复制叉重启的功能提供了线索,并为ZRANB3结构和功能的进一步研究奠定了基础。Objective The human protein ZRANB3 plays critical roles in DNA damage response pathways.To date,the structure of ZRANB3 and its functional mechanism in DNA damage response are poorly understood.To study the structure and function of ZRANB3,a protocol to produce large quantities of correctly folded andbiologically active ZRANB3 is urgently needed.The expression and purification of large and multiple-domain human proteins such as ZRANB3 are usually difficult.The objective of this study is to establish an efficient method to produce ZRANB3.Methods The ZRANB3 gene wascodon optimized for expression in E.coli,synthesized and inserted into vector pET.28a.The recombinant plasmid was transformed into E.coli Rosetta(DE3)cells and ZRANB3 expression was induced by IPTG.ZRANB3 was purified by affinity,ion exchange,gel filtration chromatography and its purity was assessed with SDS-PAGE.The biochemical function of the purified ZRANB3 was analyzed with endonuclease and replication fork reversal assays.Results This study established a protocol for the human ZRANB3 expression in E.coli and its purification.In vitro assays demonstrated that the purified recombinant ZRANB3 possesses the nuclease and replication fork regression activities reported for ZRANB3.With the established protocol,approximately 0.5 mg of biologically active ZRANB3 with greater than 95%purity can be prepared from 9 liters of E.coli culture.Conclusion This study provides an efficient method for the preparation of a large number of high-purity and enzymatically active human ZRANB3 proteins,which provides clues for a deeper understanding of its ability to promote replication fork restart,and lays a foundation for further research on the structure and function of ZRANB3.

关 键 词：ZRANB3 表达纯化 DNA损伤修复 复制叉退行 核酸内切酶 

分 类 号：Q78[生物学—分子生物学]