摘要
质粒pCE31含有在P_L起动子控制下的重组era基因,在大肠杆菌中、42℃诱导高表达出Era蛋白,经溶菌酶处理裂菌、沉淀离心洗涤后所得的纯Era是不溶的,生物活性也不高。改在40℃诱导2h,在Era底物GDP的存在下裂菌,60%以上的Era处于可溶状态。用蛋白酶抑制剂防止Era蛋白降解,经盐析、Q-SepharoseFF柱层析分离,获得可溶性纯Era蛋白。该蛋白能特异地与鸟苷酸结合、并具有GTP酶活性,表明Era是一种G蛋白。动力学定量分析结果:在4℃时,每分子Era肽链能结合一分子GTP或GDP,表明所纯化的Era蛋白几乎都具有活性;4℃下,Era蛋白与GTP或GDP结合的解离常数,分别为5.49和1.01μmol/L;37℃时,Era蛋白GTP酶的Km值为9.0μmol/L,催化GTP水解的最大速度为9.8mmolCTP/mol Era/min。
Plasmid pCE31,which contains era gene under the control of PL promoter,overproduced Era protein in E.coli after induction at 42**********C.After lyza- tion of cells by treating with lysozyme,Era protein could be obtained by washing the pellet of the lysate.However,this protein is insoluble and has only low biological activities.By changing the induction condition to 40*********C for 2 hours and adding GDP,a substrate for Era,when the cells were lyzed,over 60% of Era become soluble.Protease inhibitors were used for preventing degradation of Era.After salting out,soluble Era was purified by Q-Sepharose chromatography.The purified soluble Era bond guanine nucleotides specifically and showed CTPase activity.Kinetic analysis showed that it bond one molecule of GTP or GDP per Era peptide in vitro with a dissociation constant of 5.49 and 1.01********umol/L at 4*********C respectively.The Km of Era GTPase is 9.0*******umol/L and the maximum catalyzed rate of GTP hydrolyzed per minute per mol of Era protein at 37********C is 9.8 mmol.
关键词
ERA蛋白
基因表达
蛋白质纯化
Era protein
Gene expression,Protein purification
G protein