钙调蛋白的N末端和C末端片段载体构建和蛋白制备

Vector Construction and Protein Preparation of N-and C-terminal Lobe of Calmodulin

目的构建钙调蛋白(Ca M)的N末端片段(N-lobe)、C末端片段(C-lobe)及其钙离子(Ca2+)结合位点突变体(N-lobe12,Clobe34)原核表达载体并进行蛋白表达、纯化和活性鉴定。方法将上述c DNA片段插入PGEX-6p-3质粒载体后转化大肠杆菌BL21感受态细胞,异丙基硫代-β-D半乳糖苷(IPTG)诱导蛋白表达。利用Glutathione-Sepharose 4B beads进行分离纯化。采用SDS-PAGE检测目的蛋白纯度和相对分子质量,Bradford方法测定纯化后蛋白浓度,GST pull-down方法和膜片钳技术检测纯化后蛋白活性。结果蛋白高表达,纯化后获得高纯度、高浓度目的蛋白,纯化后蛋白能与Cav1.2型钙通道结合并可恢复已"rundown"心肌细胞膜钙通道的活性。结论本研究成功构建可以表达生物活性蛋白的N-lobe、C-lobe、N-lobe12及C-lobe34原核表达载体,为深入研究Ca M的生物学功能奠定了基础。

Objective To establish prokaryotic expression vectors for the expression, purification and activity identification of N-terminal lobe （ N- lobe ）, C-terminal lobe （ C-lobe ） of calmodulin （ CaM ） and their Ca^2＋-insensitive mutants （ N-lobe12, C-lobe34） GST fusion protein. Methods cDNAs of N-lobe, C-lobe and their mutants were individually ligated into pGEX-6p-3 plasmid vector, and then transformed into Escherichia coil BL21 component cells. The bacteria were incubated, and induced with IPTG before harvesting. The corresponding peptides were expressed as glu- tathione-S-transferase （GST） fusion proteins and purified using Glutathione Sepharose 4B beads. The GST regions of N-lobe, C-lobe and their cor- responding mutants were cleaved with PreScission Protease. SDS-PAGE was used to detect the purity and relative molecular weight. Bradford meth- od was used to determine the concentration. The protein activity was determined by pull-down assay and patch-clamp technique. Results SDS- PAGE confirmed the recombinant lobes of CaM with high purity, and high concentrations of CaM had been successfully purified. Individual N- lobe, C-lobe and their mutants could bind to N-terminal tails of Cav1.2 （NT） and rescue the channel activity from mn-down in ventricular myo- cytes of guinea-pig heart. Conclusion The prokaryotic expression vectors for the expressions of N-lobe, C-lobe and their mutants have been suc- cessfidly established, which provides the basis for further researches on biological functions of CaM.