摘要
目的:构建纤维素酶EGⅠ原核表达载体,并对表达产物进行初步酶学性质研究。方法:以康氏木霉的总RNA为模板,利用RT-PCR扩增内切葡聚糖酶EGⅠ,重组到T7启动子控制下的质粒pET.His中,构建重组质粒pET.His-EGⅠ,并将其转化至E.coli BL21(DE3)plysS感受态细胞中,经0.4mmol/L的IPTG诱导表达后对其表达产物进行13%SDS-PAGE分析。结果:SDS-PAGE电泳显示EGⅠ在大肠杆菌中得到了与预期目的蛋白相一致的外源蛋白带,分子量约45kDa。初步研究表明:重组蛋白具有内切葡聚糖酶活性,最适pH为6.0,温度在30℃～40℃时酶活稳定,金属离子Mn2+对酶活力有明显的促进作用。结论:纤维素酶EGⅠ在大肠杆菌中成功表达,具有一定活性。
Objective：To construct Prokaryotic expression vector of endoglucanaseⅠ and study the activity of the expressed production.Method：The gene coding endoglucanaseⅠ was amplified from total RNA of T.Knoningii by RT-PCR technique.The PCR product was cloned into the expression Vector pET·His which is controlled by T7promoter,and the prokaryotic expression vector pET·His-EGⅠ was thus constructed successfully.The reconstructed plasmid was transformed into E.coli BL21（DE3） plysS competent cel1.The bacterium was induced by IPTG（0.4 mmol/L）and analyzed by 13%SDS-PAGE.Result：Approximately 45kDa exogenous protein was observed on the SDS-PAGE.The result showed the enzyme optimal activity at pH 6.0 and 30℃-40℃,and the recombinant enzyme was obviously activated by Mn^2＋ ion.Conclusion：EGⅠ protein was expressed in E.coli and showed the activity,which laid a foundation of EGⅠfurther biology research.
出处
《生物技术》
CAS
CSCD
2008年第2期10-13,共4页
Biotechnology
