大肠杆菌RsmH基因克隆及表达

Gene Cloning and Expression of Escherichia Coli RsmH

以大肠杆菌全基因组DNA为模版,PCR扩增目的基因rsmh,构建pMD18-T-RsmH重组克隆载体和pET-28aRsmH重组表达载体,转化大肠杆菌BL21。经IPTG诱导表达RsmH蛋白,利用重组His-tag,通过Ni-NTA亲和层析对RsmH蛋白进行纯化。SDS-PAGE对RsmH蛋白进行定性分析,考马斯亮蓝法测定RsmH蛋白含量,确认获得质量浓度为3.91 g/L纯化的rsmh表达蛋白。

The rsmh gene sequence of E. coli was retrieved from Genebank and amplified by PCR with the genome of E. coli as template. Then rsmh was ligated to vector pMD18-T to construct pMD18-T-Rsmh. Next, expression plasmid pET-28a-RsmH was estab- lished and transformed into Escherichia coli BI21. RsmH was expressed as IPTG-inducible His-tagged recombinant enzymes and puri- fied using Ni-NTA chromatography. After purification, the RsmH protein was seperated by SDS-polyacrylamide gel electrophoresis for qualitative analysis. Protein concentration was determined with Coomassie blue method. Finally, we obtained electrophoretically pure RsmH protein of 3.91 g/L.