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1、微生物学通报SEP20,2011,38(9):1393−1399MicrobiologyChina©2011byInstituteofMicrobiology,CAStongbao@im.ac.cn研究报告大肠杆菌外膜蛋白酶T及其突变体的表达、复性及生物活性分析1111121*刘晓露惠长野赵铁彭亮张文炳黄胜和曹虹(1.南方医科大学公共卫生与热带医学学院微生物学系广东广州510515)(2.南加州大学洛杉矶儿童医院美国洛杉矶90027)摘要:外膜蛋白酶T(Outer-membraneproteaseT,OmpT
2、)是定位于大肠杆菌外膜,具有高度底物特异性的蛋白水解酶。本文旨在建立克隆表达膜蛋白OmpT和体外复性的方法,考察其蛋白酶活性。首先以大肠杆菌基因组DNA为模板,PCR扩增ompT基因,连接至pET28a(pET-ompT),引入点突变Asp85Ala,构建表达质粒pET-ompT85。然后将两种重组质粒转化入BL21(DE3),均以包涵体形式大量表达。纯化后的蛋白经稀释法复性,并加入粗制脂多糖(Lipopolysaccharide,LPS)恢复蛋白酶活性。通过SDS-PAGE、鱼精蛋白水解试验及生长曲线观察表
3、明,重组蛋白OmpT在体外能水解抗菌肽鱼精蛋白和兔肌肉肌酸激酶,而OmpT突变体则无上述功能。上述结果表明本文获得了具有蛋白水解酶功能的重组蛋白OmpT,该蛋白在体外可保护大肠杆菌抵抗鱼精蛋白的杀菌作用。关键词:大肠杆菌,外膜蛋白T,突变体,生物活性Expression,refoldingandcharacterizationofEscherichiacolioutermembraneTanditsmutant11111LIUXiao-LuHUIChang-YeZHAOTiePENGLiangZHANGWen
4、-Bing21*HUANGSheng-HeCAOHong(1.DepartmentofMicrobiology,SchoolofPublicHealthandTropicalMedicine,SouthernMedicalUniversity,Guangzhou,Guangdong510515,China)(2.Children’sHospitalLosAngeles,UniversityofSouthernCalifornia,LosAngeles,CA90027,USA)Abstract:OmpT,loc
5、atedinEscherichiacoli(E.coli)outermembrane,isaproteasethatdemonstrateshighlysubstratespecificity.Inordertoestalishtheapproachesforexpressionandrefoldingofmem-braneproteinOmpT,andexaminethedemonstratedproteaseactivityofOmpT,theompTgenewasfirstamplifiedbyPCRa
6、ndinsertedintopET28a(pET-ompT)andintroducedbyAsp85Alasite-directed基金项目:国家自然科学基金项目(No.30972637)*通讯作者:Tel:86-20-61648723;Fax:86-20-61648307;:gzhcao@fimmu.com收稿日期:2011-03-11;接受日期:2011-06-011394微生物学通报2011,Vol.38,No.9mutagenesistogeneratemutantAsp85Ala(pET-ompT8
7、5).Then,thetworecombinantplasmidsweretransformedintoBL21(DE3),OmpTandthemutantwereexpressedintheformofinclusionbodies,purifiedandrefoldedbyN-Dodecyl-N,N-dimethyl-1-ammonio-3-propanesulphonate.Theadditionoflipopolysaccharide(LPS)totherecombinantOmpTwascritic
8、altorefolditsproteaseactivityinvitro.Finally,theabilityoftherecombinantwide-typeOmpTtohydrolyzeprotamineandrabbitmusclecreatinekinase(RMCK)wasconfirmedbySDS-PAGE,bacteriaagglutinationandgrowthcurveinco