人La蛋白突变体表达质粒的构建及诱导表达纯化

目的:构建人La蛋白(human La protein,hLa)突变体表达质粒,并在大肠杆菌中表达野生型La蛋白及各突变体.方法:利用定点突变技术,对野生型人La蛋白的原核表达质粒pET28b-hLa进行定向缺失突变,将得到的3个突变体Mu1(A366)、Del1(A235～276)、Del2(A119～150)分别克隆至pET28b中,获得野生型人La蛋白突变体的表达质粒,并在BL21(DE3)和Rosetta2两种不同宿主菌中和不同浓度的IPTG诱导条件下进行蛋白表达水平的比较.结果:所获得的人La蛋白突变体的基因编码序列经测序符合序列设计的要求,表达产物经SDS-PAGE分析可见,在相对分子质量47 000处出现一明显条带,与预期的相对分子质量一致.结论:三个位点的序列改变并没有明显影响hLa蛋白的表达,在补充密码子的宿主菌Rosetta2中的表达量优于宿主菌BL21(DE3).

Construction of human La protein mutant plasmids and expression,purification of human La protein and its mutants

Objective:To construct mutant plasmids of the human La protein and express human La protein and its mutants in E. coli. Methods: The prokaryotic expression plasmid pET28b-hLa was deleted at different sites (Mu1A366],Del1A235-276],Del2A119-150]) using site-directed mutation technique. The PCR products of the mutant clones were digested with appropriate restriction enzyme and were inserted into prokaryotic expression vector pET28b. The recombinant mutants plasmids were transformed into different E.coli strains: BL21(DE3) and Rosetta2. The human La protein and its mutants were induced by different concentrations of IPTG to get the optimal condition for protein expression. Results: Sequencing showed that La mutants gene obtained in this study met the designed requirements. SDS-PAGE and Western blot analysis showed that the relative molecular weight of La protein and its mutants was about 47 000,which was accordant with that expected. Conclusion: The expression vector of La protein mutants has been successfully obtained and expressed in Rosetta2.