重组钙调蛋白及其突变体蛋白的分离纯化及活性鉴定

目的建立一种简单稳定高效分离纯化体外重组钙调蛋白(CaM)及其突变体蛋白的方法。方法将CaM及其三种钙结合位点突变体的cDNA插入pGEX-6p-3质粒载体后,转化大肠杆菌BL21感受态细胞,大量培养并利用异丙基硫代-β-D半乳糖苷(IPTG)诱导CaM及其突变体的GST融合蛋白表达,GS-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;采用Bradford方法测定纯化后蛋白浓度;采用膜片钳技术检测纯化后蛋白的活性。结果纯化的CaM及突变体蛋白具有较高的纯度;CaM及突变体蛋白得到了大量表达;纯化后蛋白可恢复已"run-down"心肌细胞膜钙通道的活性。结论本研究成功建立了一种稳定高效简单的重组CaM及其突变体蛋白的分离纯化方法,为深入研究CaM的生物学功能奠定了基础。

Purification and identification of recombinant calmodulin and mutant

Purpose To establish a stable,rapid and effective method for the purification of calmodulin(CaM)and its mutants.Methods Escherichia coli BL21 competent cells were transformed with pGEX-6p-3 plasmid vector,which was inserted with the cDNAs of CaM and its mutants.The transformed BL21 cells were incubated,and stimulated with IPTG to express GST fusion proteins of CaM and its mutants.The fusion proteins were purified with Glutathione-Sepharose 4B beads.SDS-PAGE was used to detect the purity and relative molecular weight of CaM and its mutants.Bradford method was used to determine the concentration of purification CaM and its mutants,and the plasmids made a sequence analysis.The protein activity was examined by patch-clamp technique.Results SDS-PAGE showed the high purity of CaM and its mutants.The concentration of CaM and its mutants was around 1 mg/mL,which was enough for molecular biological and electrophysiological studies.The CaM and its mutants purified by this method could reverse run-down of L-type Ca2+ of guinea pig cardiac myocytes.Conclusion A stable,rapid and effective method for the purification of calmodulin and its mutants was found,which provided the basis for further researches on CaM′s biological functions.