重组人PRL-3蛋白的表达、鉴定和酶动力学分析  

Expression,identification and enzyme kinetics analysis of recombinant human PRL-3 protein

作　　者：何太平[1] 梁念慈[2] 

机构地区：[1]广东医学院营养与食品卫生学教研室,广东东莞523808 [2]广东天然药物研究与开发重点实验室,广东湛江524023

出　　处：《广东医学院学报》2011年第4期359-363,共5页Journal of Guangdong Medical College

基　　金：广东省科技计划项目(No.2007B030702003)

摘　　要：目的构建融合蛋白pColdII-His-PRL-3表达质粒,并进行诱导表达、纯化、鉴定和酶动力学分析。方法 2+通过基因重组技术构建pColdII-His-PRL-3融合蛋白表达载体,并在ArcticExpress E.Coli中表达融合蛋白。经Ni-NTA层析纯化,PRL-3蛋白用SDS-PAGE和Western blot初步鉴定,基质辅助激光解吸/电离直角型飞行时间质谱(MALDI-TOF-TOF)确证,并进行酶动力学分析。结果重组载体pColdII-His-PRL-3包含正确的PRL-3编码序列,经IPTG诱导后能正确表达融合蛋白,Westernblot显示表达蛋白具有抗原性,质谱分析结果显示目的蛋白氨基酸序列与-1PRL-3蛋白完全相符。PRL-3酶的米氏常数Km为6.59μmol/L,催化常数Kcat为0.44min。结论构建的pColdII-His-PRL-3重组载体可表达具有酶活性的PRL-3蛋白,为PRL-3酶抑制剂的筛选奠定了基础。Objective To study the expression,identification and enzyme kinetics of pCold II-His-PRL-3 expression vector.Methods pCold II-His-PRL-3 expression vector was constructed and then transfected into ArcticExpress E.coli.Expression of His-PRL-3 fusion protein was induced by IPTG.After purified by Ni2＋-NTA resin,the purified protein was verified with SDS-PAGE,Western blot,MALDI-TOF-TOF and enzyme kinetics analysis.Results The pCold II-His-PRL-3 expression vector included right coding sequence of PRL-3.The fusion protein was expressed in ArcticExpress E.coli after IPTG induction,and had antigenicity using Western blot.Mass chromatographic analysis showed that the amino acid sequence of target protein was consistent with that of PRL-3 protein.Michaelis constant（Km） and catalytic constant（Kcat） of PRL-3 enzyme were 6.59 μmol/L and 0.44 min-1,respectively.Conclusion The constructed pCold II-His-PRL-3 expression vector can express PRL-3 protein with enzyme activity,which provides the foundation for screening PRL-3 enzyme inhibitor.

关 键 词：PRL-3 蛋白表达 酶动力学 

分 类 号：Q784[生物学—分子生物学]