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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。
Objective To establish a simple, stable and efficient method for the isolation and purification of recombinant calmodulin (CaM) and its mutant protein in vitro. Methods The cDNA of CaM and its three calcium-binding site mutants was inserted into pGEX-6p-3 plasmid vector and then transformed into E. coli BL21 competent cells. After extensive culture and using isopropylthio-β-D galactoside IPTG) induced GST fusion protein expression of CaM and its mutant, GS-4B beads were isolated and purified. Purity and relative molecular mass of the target protein were determined by SDS-PAGE. The purified protein concentration was determined by Bradford method. The purified protein was tested by patch-clamp technique. Results The purity of purified CaM and mutant protein was high. The expression of CaM and mutant protein was abundant. After purification, the protein could restore the activity of calcium channel in myocardial cell membrane. Conclusion This study successfully established a stable and efficient method for the isolation and purification of recombinant CaM and its mutant protein, which lays the foundation for further study on the biological function of CaM.