肌球蛋白轻链激酶CaM结合位点突变体的构建

Construction of Calmodulin-binding site mutant of myosin light chain kinase

目的:平滑肌肌球蛋白轻链激酶(myosin light chain kinase,MLCK)具有激酶活性和非激酶活性,在平滑肌收缩过程中起着关键酶调控的作用。为探寻MLCK的非激酶活性区域对MLCK活性的影响,本实验利用分子生物学技术构建了肌球蛋白轻链激酶CaM结合位点突变体,并纯化出重组的MLCK表达的蛋白质,为深入研究MLCK的非激酶活性在调节平滑肌收缩过程中的分子机制提供了实验基础。方法:利用野生型MLCK全长的cDNA序列设计CaM结合位点的突变引物,利用PCR技术进行定点突变,获得CaM结合位点的突变体(△CaM/MLCK)。在大肠杆菌中表达重组CaM结合位点的突变体(△CaM/MLCK),通过亲和层析及凝胶过滤进行分离纯化重组蛋白,SDS-PAGE检测表达及纯化的重组蛋白。结果:构建重组MLCK钙调蛋白结合位点突变体(△CaM/MLCK),△CaM/MLCK在大肠杆菌中以可溶形式大量表达并得到纯化。结论:成功构建重组MLCK钙调蛋白结合位点突变体(△CaM/MLCK)并获得纯化的表达蛋白质。

Objective： Myosin light chain kinase （MLCK） possesses kinase and non-kinase activities, and both of them play the important roles in the regulation of smooth muscle contraction. To explore the non-kinase activity of MLCK, we constructed the MLCK CaM-binding site mutant, and obtained the recombinant mutant of MLCK. Methods： We designed CaM-binding site mutant primers, and PCR is used to site-directed mutagenesis by using wild-type MLCK,cDNA as the template. CaM-binding site mutant MLCK （△CaM/MLCK） are recombinated and expressed in E. coli, affinity chromatography and gel filtration are used for the separation and purification of expressed protein. SDS-PAGE was used to detect expression and purification of recombinant protein. Results： The recombinant of MLCK calmodulin-binding site mutant （△CaM/MLCK） is constructed, and △CaM/MLCK was expressed in Escherichia coli as a soluble form. Conclusion： The construction and expression of recombinant MLCK calmodulin-binding site mutant （△CaM / MLCK） are successfully established.