变形链球菌ComE蛋白在大肠杆菌中的高效表达与纯化

High-level expression and purification of ComE in Escherichia coli

目的:构建变形链球菌comE基因原核表达载体,诱导ComE融合蛋白高效表达,获取高质量的ComE融合蛋白。方法:提取变形链球菌基因组,PCR扩增得到变形链球菌comE基因片段,BamH Ⅰ和Xho Ⅰ双酶切后定向克隆到pET28a(+)表达载体中,并转化入E.coli BL21(DE3)。IPTG诱导融合蛋白表达,Western blot鉴定表达产物。进行蛋白表达的可溶性鉴定,并优化诱导时菌液的A600值、IPTG浓度、诱导时间和温度4个表达条件。然后用优化的条件大量诱导表达ComE融合蛋白,采用镍柱和分子筛两步法进行纯化。结果:经PCR、双酶切反应以及测序鉴定,重组质粒中的插入序列为comE基因的全长片段(753bp),无碱基的突变与读码框架的偏移。Western blot证实表达产物确为6×His-ComE融合蛋白,相对分子质量约为33000。目的蛋白在上清和沉淀中均有表达,且当A600为0.4时,加入IPTG至终浓度0.10mmol/L,37℃诱导4h后表达量最大。结论:成功构建pET28a(+)-comE原核表达载体,用优化后的6×His-ComE融合蛋白在E.coli BL21(DE3)中的表达条件,获得纯化的变形链球菌ComE融合蛋白。

Objective： To construct the prokaryotic expression vector pET28 a （＋） - comE, to get the highest expression of comE in E. coli BL21 （DE3） , and to obtain the purified ComE fusion protein. Methods： ComE gene was amplified by PCR with specific primers from genome of Streptococcus matans UA159. After digested by two restriction endonucleases BamH Ⅰ and Xho Ⅰ , the gene segment was inserted into vector pET28a（＋）. Then the recombinant vector was transformed into E. coli BL21 （ DE3 ）. The protein expression was induced by IPTG and the result was confirmed by Western blot. The solubility of the fusion protein was examined by 12% SDS-PAGE, and the expression conditions were optimized. Finally the fusion protein was purified by a two-step purification procedure utilizing Ni^2 ＋ chelating column and S75 size exclusion column. Results ： The recombinant plasmid was confirmed by PCR, restriction endonucleases digestion and sequence analysis, which showed that the inserted segment was identical to comE gene reported by GenBank without nucleotide mutation. The fusion protein which was confirmed by Western blot can be expressed in E. coli BL21 （ DE3 ） and existed both in supernatant and precipitation. The maximum expression product amount was obtained when the A600 value of E. coil BL21 （DE3） was 0.4, IPTG concentration was 0.10 mmol/L and induction time was 4 h. Conclusion： The recombinant plasmid is constructed and the ComE fusion protein is purified by the optimum conditions.