细菌外膜蛋白BamA突变体构建及位点运动性分析  

Construction and motility analysis of residue sites of bacterial outer membrane protein BamA mutants

作　　者：王媛 李志慧 魏倩 董国福 储引娣 王长振 WANG Yuan;LI Zhi-hui;WEI Qian;DONG Guo-fu;Chu Yin-di;WANG Chang-zhen(Institute of Radiation Medicine,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100850,China;State Key Laboratory of Medical Molecular Biology,Department of Microbiology and Parasitology,Institute of Basic Medical Sciences,Chinese Academy of Medical Sciences,School of Basic Medicine,Peking Union Medical College,Beijing 100005,China)

机构地区：[1]军事科学院军事医学研究院辐射医学研究所,北京100850 [2]中国医学科学院基础医学研究所北京协和医学院基础学院病原学系,医学分子生物学国家重点实验室,北京100005

出　　处：《军事医学》2022年第9期677-681,共5页Military Medical Sciences

基　　金：国家自然科学基金(31800053)。

摘　　要：目的构建细菌外膜β桶状蛋白组装机器(BAM)核心组分BamA点突变原核表达载体,获得特定位点突变BamA蛋白,并分析该蛋白多肽易位相关(POTRA)结构域上G313位点运动特性。方法利用重叠延伸PCR(SOE-PCR)将BamA蛋白第690和700位天然半胱氨酸(C)突变为丝氨酸(S),将POTRA结构域上第313位氨基酸由甘氨酸(G)突为C,构建含BamA-C690S-C700S-G313C突变的原核表达载体pET22b-Strep-BamA。异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导突变蛋白表达后,利用Strep-Tactin Sepharose柱亲和层析方法对突变体蛋白进行纯化。甲烷硫代磺酸(MTSL)标记所得纯化蛋白的半胱氨酸后,进行电子顺磁共振(EPR)波谱检测,通过EPR波谱拟合获得旋转相关时间τ_(c),分析G313C氨基酸位点的运动特性。结果构建了BamA-C690S-C700S-G313C突变体原核表达载体,实现了突变体蛋白有效表达和纯化。G313C位点EPR波谱为单成分运动状态波谱,其τc值为(2.30±0.03)ns。结论该研究建立了BamA点突变原核表达和蛋白纯化方法,并获得BamA蛋白G313C运动性特征参数,为进一步研究BamA蛋白在外膜蛋白整合过程中的结构机制提供了数据。Objective To construct a prokaryotic expression vector ofβ-barrel assembly machinery A(BamA)mutants,and analyze the mobility of residue site glycine(G)313 in the polypeptide transport-associated domain(POTRA)of BamA.Methods Site-directed mutagenesis was performed using sequence overlapped extension PCR(SOE-PCR).Two natural cysteines at sites 690 and 700 were mutated to serine(S)while the glycine(G)at site 313 in the POTRA domain was mutated to cycteine(C)so that the prokaryotic expression vector pET22b-Strep-BamA containing C690S-C700SG313C was constructed.The BamA mutants were induced with isopropylβ-D-thiogagactopyranosid(IPTG)and the expression product was purfied using Strep-Tactin Sepharose.The cysteine was labeled with 2,2,5,5-tetramethyl-1-oxyl-3-methyl methanethiosulfonate(MTSL)for electron paramagnetic resonance(EPR)experiments.The rotational correlation timeτcof the EPR spectrum was calculated in order to analyze the mobility characteristics of G313C amino acid.Results The prokaryotic expression vector pET22b-Strep-BamA containing mutated sites was constructed.The mutant protein was efficiently expressed and purified.The EPR spectrum of G313C site presented as a single-component spectrum.The τ_(c) value of G313C site was calculated as(2.30±0.03)ns.Conclusion The BamA recombinant mutant protein is expressed and purified in a prokaryotic expression system.The results reveal the mobility characteristics of G313C amino acid site and provide data for future studies on the structural mechanism of BamA in the process of outer membrane protein(OMP)integration.

关 键 词：细菌外膜蛋白质类 BamA 突变 定点自旋标记-电子顺磁共振 运动性 

分 类 号：R378[医药卫生—病原生物学]