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

作　　者：魏晓晴[1] 张月[2] 赵莹[1] 吕广艳[1] 崔颖[1] 王辉[1] 高颖[1,2] 

机构地区：[1]辽宁省医学细胞分子生物学重点实验室 [2]大连医科大学生化教研室,辽宁大连116044

出　　处：《现代生物医学进展》2009年第17期3247-3250,共4页Progress in Modern Biomedicine

基　　金：国家自然科学基金资助项目(30470394)

摘　　要：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.

关 键 词：肌球蛋白轻链激酶 钙调蛋白 突变体 

分 类 号：Q75[生物学—分子生物学] Q78