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目的探讨心肌Cav1.2钙通道NT蛋白片段提取与纯化的方法,并研究其与CaM的相互作用。方法将NT的cDNA插入pGEX-6p-3质粒载体后,转化大肠杆菌BL-21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NT的GST融合蛋白表达,GS-4B beads分离纯化,Pre Scission蛋白酶切除GST标签,进行十二烷基硫酸钠-聚丙烯酰氨凝胶电泳(SDSPAGE)检测蛋白纯度和相对分子质量。采用GST pull-down实验研究NT片段与Ca M的相互作用。结果 SDS-PAGE结果显示成功纯化获得NT蛋白片段,且具有较高的纯度。GST pull-down实验结果显示NT可与Ca M结合,且具有浓度依赖性。结论本研究成功制备与纯化了心肌Cav1.2钙通道NT蛋白片段,为深入研究蛋白相互作用及NT的生物学功能奠定了基础。
Objective To investigate the method of extraction and purification of myocardial Cav1.2 calcium channel NT protein fragment and study its interaction with CaM. Methods The cDNA of NT was inserted into pGEX-6p-3 plasmid vector and then transformed into E. coli BL-21 competent cells. A large amount of NTT-induced GST fusion protein was induced by isopropyl thio-β-D-galactoside The purity of GS-4B beads and the molecular weight of GS-4B beads were determined by SDS-PAGE. The GST-tag was excised from PreScission protease and the protein purity and relative molecular mass were determined by SDS-PAGE. The GST pull-down experiment was used to investigate the interaction of NT fragment with CaM. Results The results of SDS-PAGE showed that NT protein fragment was successfully purified and had high purity. GST pull-down results show that NT can bind to CaM in a concentration-dependent manner. Conclusion This study successfully prepared and purified myocardial Cav1.2 calcium channel NT protein fragment, which laid the foundation for further study of protein interaction and the biological function of NT.