人钙调素在大肠杆菌中的表达、纯化及其活性研究

利用基因重组技术,将经PCR扩增获得的人钙调素基因(hCaMcDNA)插入质粒pBV220,构建重组表达载体hCaM/pBV220,用酶切、DNA测序、PCR扩增鉴定阳性克隆.阳性重组子在大肠杆菌DH5α中经温度诱导可高效表达CaM蛋白,经15%SDS-PAGE分析,可观察到一与CaM分子量相符(约17kD)的诱导表达条带,其表达量占菌体蛋白总量20%,并主要以可溶性形式表达.Westernblot结果证实,17kD的表达条带可与标准鼠抗人CaM单克隆抗体起特异反应.用Pheny1-SepharoseCL-4B疏水亲和层析法纯化重组菌超声上清表达产物,每1L菌液可获CaM纯品3～4mg.重组人CaM(rhCaM)与牛脑CaM的氨基酸组成基本一致.生物活性测定结果提示,rhCaM具有激活NAD激酶的活性,其激活程度与标准人脑CaM几乎一致.

Expression and Purification of Human Calmodulin in E.coli and Estimation of Its Biological Activity

The gene coding for human CaM was amplified by PCR in which pUC/hCaMⅢ cDNA was used as template. After inserting the hCaMⅢ cDNA into the expression plasmid pBV220, the hCaMⅢ cDNA recombinant expression vector (hCaMⅢ/pBV220) was constructed which was confirmed by DNA sequencing, restriction enzyme digestion and PCR identifications. The recombinant plasmid was transformed into E. coli DH5 α. After heat induction of the bacterial culture transformed by the recombinant plasmid, a high level expression of CaM protein was obtained. SDS PAGE analysis showed that the recombinant E. coli could express a 17 kD protein which accounted for about 20% of the total cellular protein. The solubility of expression protein indicated that CaM was expressed predominantly in the soluble form. Western blot analysis showed that anti CaM McAb specifically bound to the 17 kD band of expression product. rhCaM was purified by Phenyl Sepharose CL 4B affinity chromatography from recombinant bacterial lysate.3～4 milligrams of the purified protein were obtained from 1 L of bacterial culture. The amino acid compositions of rhCaM were identical with those of bovine brain CaM. The rhCaM was able to activate NAD kinase to the same extent as the standard human brain CaM.