ΦC31位点特异性整合酶DNA结合功能域的鉴定  

Truncation Analysis of Phage ΦC31 Integrase Revealing the Potential DNA Binding Domains

作　　者：辛清婷[1] 马金芳[1] 王玮[1] 刘绍辉[1] 李荣秀[2] 朱焕章[1] 

机构地区：[1]复旦大学生命科学学院遗传学研究所遗传工程国家重点实验室,上海200433 [2]上海交通大学生命科学技术学院微生物代谢教育部重点实验室,上海200240

出　　处：《中国生物化学与分子生物学报》2010年第7期622-629,共8页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金(No30671190;No3071120569);国家科技重大专项项目(No2008ZX10001006)~~

摘　　要：DNA结合功能域的确定是阐明位点特异性重组酶整合机制的关键,而对酶DNA结合功能域进行突变研究是提高酶整合效率和整合特异性的重要方法.为了鉴定ΦC31位点特异性整合酶的DNA结合功能域,依据对ΦC31整合酶序列的生物信息学分析结果,利用PCR和克隆技术在pET22b原核表达载体上构建ΦC31整合酶重组截短突变体表达质粒,将获得的表达质粒转化入大肠杆菌BL21(DE3)菌株扩大培养并用IPTG诱导融合蛋白的表达,经镍柱纯化获得了纯度达90%以上的重组蛋白,分子量也与预期大小一致,Western印迹确定了重组蛋白的特异性.凝胶迁移滞后实验显示野生型以及截短突变体蛋白ΦC311-528、ΦC311-472、ΦC311-413能与细菌附着位点DNAattB和噬菌体附着位点DNAattP结合的条带,而截短突变体ΦC311-353、ΦC311-279、ΦC311-120观察不到相应的结合条带.6个截短突变体质粒在体内重组活性蓝白斑实验中均表现为蓝斑,显示出皆丧失体内重组活性.研究证实,ΦC31整合酶半胱氨酸富集域(第353～413位氨基酸)具有DNA结合的功能,而C末端缬氨酸富集区(第528～613位氨基酸)也与其重组活性相关.这为进一步了解ΦC31整合酶的结构与功能,最终引导其结构进化,提高其特异性和整合效率奠定了基础.Identifying the DNA binding domains is the key to clarify the mechanism of site-specific recombinase,and the mutation research in the domain could help to improve the integration efficiency and specificity.In order to identify the DNA binding domains of ΦC31 integrase,we constructed six truncated mutants based on the structure prediction：the cDNA sequences encoding six truncated mutants of ΦC31 integrase were amplified and subcloned into the pET22b vector to construct the prokaryotic expression plasmids.The six recombinant plasmids were transformed into E.coli BL21（DE3） strain,respectively,and then the exogenous protein expression was induced by IPTG.After purification with Ni-NTA,more than 90% purity of recombinant protein was obtained,the molecular weight was also consistent with the expectation,and the specificity was confirmed by Western blotting assay.Electrophoretic mobility shift assay（EMSA） showed that mutants ΦC311-528,ΦC311-472 and ΦC311-413 could bind to both attB and attP,while,mutants ΦC311-353,ΦC311-279 and ΦC311-120 were unable to bind to the specific target DNA with the same efficiency as the wild-type integrase.Furthermore,all derivatives completely lost their recombination activity,as evidenced by recombination assay in E.coli.These data reveal that the Cys-rich region（aa 353～413） is essential to DNA binding,and the Cterminus（aa 528 ～ 613） may play a crucial role in affecting the recombination activity.This research is useful in understanding the relationship between the structure and function,which would further help to improve the efficiency and specificity of ΦC31 integrase.

关 键 词：ΦC31整合酶 截短型突变体 原核表达和纯化 DNA结合能力 

分 类 号：Q51[生物学—生物化学] Q78