限制性蛋白酶解提高E.coli Hfq蛋白晶体结构分辨率  

Limited Proteolysis Improves E. coli Hfq Crystal Structure Resolution

作　　者：冯是琼 司云龙 宋晨阳[1] 王佩琦[1] 苏纪勇[1] 

机构地区：[1]东北师范大学生命科学学院生物化学与分子生物学系,长春130024

出　　处：《中国生物化学与分子生物学报》2015年第10期1102-1108,共7页Chinese Journal of Biochemistry and Molecular Biology

基　　金：中央高校基本科研业务费专项资金资助(No.2412015KJ018)~~

摘　　要：Hfq蛋白是一种促进sRNA结合互补RNA的细菌伴侣蛋白.Hfq蛋白可以抑制或上调目的蛋白的表达及促进mRNA的降解,从而调控细菌的生理功能使其适应周围胁迫环境.Hfq是同源六聚体蛋白质,在其中心部位有1个内环,每个单体上有1个极其保守的组氨酸分布在这个内环表面.根据Hfq这个特点,应用镍离子亲和柱直接从大肠杆菌中纯化Hfq,接着用凝胶过滤层析柱纯化,最终获得了纯度70%的Hfq蛋白.对该蛋白质进行结晶条件初筛前,应用胰凝乳蛋白酶对其进行限制性蛋白酶解,切除其C端无规则卷曲,便于Hfq结晶及提高晶体衍射率.初期得到的Hfq晶体是极小的针状晶体,通过优化结晶条件获得了较大的晶体.最终解析出了2个分辨率较高的Hfq晶体结构,其中1个晶体结构分辨率达到了1.63.Hfq is an abundant RNA chaperone in bacteria. Hfq facilitates sRNAs targeting their antisense RNAs that regulates bacteria responses in stressing environments. Hfq is a homohexameric protein which has six histidine residues on the surface of its inner ring. Therefore,nickel affinity chromatography can be used to directly purify this protein from E. coli. After size exclusion chromatography,the purity of this protein achieved 70%. Before crystallization,this protein was limited proteolysised by chymotrypsin to cut C-terminal flexible loop. The digested Hfq fragment was crystallized in many conditions containing（ NH4）2SO4as precipitant. But the initial needle-like crystals are too small to be used for crystal data collection. After crystallization optimization,bigger crystals grew in two conditions. Finally,two Hfq crystal structures were solved. The resolution of one crystal structure was 1. 63 .

关 键 词：限制性蛋白酶解 蛋白质晶体结构分辨率 蛋白质纯化 蛋白质结晶优化 

分 类 号：Q5[生物学—生物化学]