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中文摘要：
目的 构建钙调蛋白（CaM）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-lobe，C-lobe34）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 cDNA 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“run- down”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 CaM 的生物学功能奠定了基础。
英文摘要：
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.
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