肺炎克雷伯菌CRP蛋白表达与纯化及生物学特征分析

目的:原核表达获得肺炎克雷伯菌KP1_RS23625基因（crp）编码的CRP蛋白，并解析CRP蛋白具体的生物学功能。方法:首先克隆肺炎克雷伯菌crp基因，并亚克隆至pET-28a（+）质粒构建重组蛋白表达载体；然后体外原核诱导表达、纯化目的蛋白并进行SDS-PAGE电泳分析。进一步通过生物学信息方法分析CRP蛋白的生物学功能:利用ProtParam、Protscale、SignalP4.1 Service、TMHMM Server v.2.0、SOPMA、SWISS-MODEL软件分别分析目的蛋白理化性质、疏水性、信号肽、跨膜区域、二级与三级结构等一般生物学特征；并借助CDD数据库、Net Phos 3.1 Sever在线软件预测蛋白的功能结构域、磷酸化位点等功能特征；进一步利用ATRING 11.0交互式数据库进行蛋白质交联互作分析。结果:成功构建了重组目的蛋白表达载体pET-28a-crp，并通过原核诱导表达与镍柱亲和吸附法表达、纯化获得CRP目的蛋白；蛋白电泳结果显示CRP蛋白为水溶性蛋白，包括标签蛋白（约4 kDa）的重组目的蛋白分子质量约27 kDa。对CRP蛋白的生物信息学分析结果显示:CRP蛋白大小23.65 kDa、为亲水性蛋白；无跨膜结构域、无信号肽；蛋白二级结构主要由α螺旋与不规则卷曲构成，其中α-螺旋占比40%以上，结构较松散；同时成功构建了三级结构模型；CRP蛋白结构域分析表明含有1个功能结构域，属PRK11753超级家族；修饰位点及蛋白注释分析显示目的蛋白含20个磷酸化位点，与多个蛋白具有交互作用。结论:本研究利用基因工程方法成功获得了较高纯度肺炎克雷伯菌CRP蛋白，并通过生物信息学手段解析了其生物学特征，为肺炎克雷伯菌感染的临床治疗提供理论基础。

Expression,Purification and Analysis of the Biological Characteristics of Klebsiella pneumoniae CRP Protein

Objective:This study aimed to prokaryotically express and purify the protein encoded by KP1_RS23625 gene(crp)of Klebsiella pneumoniae in vitro,and analyze the biological function of CRP protein by means of biological information.Methods:CRP gene was amplified and cloned,and then subcloned into pET-28a(+)plasmid to construct a recombinant expression vector.In vitro induced expression,purification of the target protein were done,and analyzed by SDS-PAGE electrophoresis.Furthermore,the biological function of CRP protein was analyzed using several biological information softwares:The general biological characteristics of the target protein including physicochemical properties,hydrophobicity,signal peptide,transmembrane region,secondary and tertiary structure were analyzed via ProtParam,Protscale,SignalP4.1 Service,TMHMM Server v.2.0,SOPMA and SWISS-MODEL softwares respectively,the functional characteristics of domain and phosphorylation site were predicted by the CDD database and online Net Phos 3.1 Sever software,respectively,and protein crosslink interaction was performed through ATRING 11.0 interactive database.Results:The recombinant target protein expression vector pET-28a-crp was successfully constructed,and the CRP target protein was obtained by prokaryotic induction expression and nickel column affinity absorption method,and the electrophoresis results showed that the CRP protein is a water-soluble protein,and the molecular weight of the recombinant protein including the tag protein(4 kDa)was about 27 kDa.Bioinformatics analysis showed that CRP protein had a size of 23.65 kDa,which was a hydrophilic protein and consistent with electrophoresis results,and had no transmembrane domain,no signal peptide.The secondary structure was mainly composed ofα-helix and irregular coils,of whichα-helix accounts for more than 40%and the structure was loose.The tertiary structure model was successfully constructed.The analysis of CRP protein domain indicated that it contained one functional domain,belonging to the PRK11753 super family,the modification site and annotation analysis showed that the target protein contains 20 phosphorylation sites,which could interact with multiple proteins.Conclusion:In this study,Klebsiella pneumoniae CRP protein with a higher purity was successfully obtained by genetic engineering methods and the biological characteristics were revealed,which could provide basic support for the clinical treatment of K.pneumoniae infection.