重组平滑肌肌球蛋白轻链激酶及ATP结合位点突变体的构建及表达

Construction and Expression of the Recombinant Smooth Muscle Myosin Light Chain Kinase and the ATP Binding Site Mutant

目的构建重组全长平滑肌肌球蛋白轻链激酶(myosin light chain kinase,MLCK)ATP结合位点突变体,以研究平滑肌MLCK的结构与功能。方法利用试剂盒对MLCKATP结合位点进行定点突变,构建全长平滑肌MLCKATP结合位点突变体重组表达载体pCold/BsMLCK/△ATP,在大肠杆菌中表达;利用SDS-PAGE鉴定表达的重组全长平滑肌MLCKATP结合位点突变体在细胞中的分布;运用亲和层析及凝胶过滤分离纯化重组全长MLCK并利用SDS-PAGE鉴定表达MLCK的纯度。结果重组全长MLCK/△ATP(突变型)在大肠杆菌中以可溶的形式大量表达;在样品的上清和沉淀中均有重组全长MLCK/△ATP表达;经CaM-Sepharose4B和Superose6HR纯化,SDS-PAGE鉴定得到单一的表达条带。结论重组全长MLCK/△ATP(突变型)可以在大肠杆菌中以可溶形式大量表达;SDS-PAGE结果显示重组全长MLCK/△ATP可以通过亲和层析和凝胶过滤纯化得到单一的条带。

Objective To construct the ATP binding site mutant of the recombinant smooth muscle myosinlight chain kinase（MLCK）, and study the form and the function of MLCK furthermore. Methods We explored the site-directed mutation technique to mutate the ATP binding sites of MLCK. The recombinant ATP binding site mutants expression vector （pCold/BsMLCK/△ATP） was expressed in E.coli. Using SDS-PAGE to confirm the distribution of the recombinant MLCK/△ATP in cells and using affinity chormatography and gel filtration to purify recombinant MLCK/△ATP. We indentificated the purity the MLCK/AATP with SDS-PAGE. Results the ATP binding site mutagenesis of MLCK （MLCK/△ATP） expressed in E.coli. the MLCK/A ATP existed in both supernatant and precipitate of the sample.Purified MLCK showed one single band in SDS-PAGE using the CaM- Sepharose 4B and Superose 6 HR purification system. Conclusions 1.the ATP binding site mutagenesis of MLCK （MLCK/△ATP） expressed in E.coli with a large amount and existed as a soluble form 2. SDS-PAGE analysis showed that the recombinant MLCK/△ATP can be purified by affinity chromatography and gel filtration..