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目的探讨心肌Ca_V1.2钙通道CT3蛋白片段提取与纯化的方法。方法将CT3的cDNA插入pG EX-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CT3的GST融合蛋白表达,并采用超声破碎法进行分离纯化,在10mmol/L二硫苏糖醇(DL-dithiothreitol,DTT)存在的情况下,PreScission蛋白酶切除GST标签,凝胶电泳鉴定CT3蛋白,检测蛋白纯度和相对分子质量。结果 SDSPAGE结果显示,超声破碎法可以纯化得到GST-CT3融合蛋白,且单纯应用PreScission蛋白酶酶切GST-CT3,得到的CT3蛋白片段浓度较低,而采用PreS cission蛋白酶联合应用DTT则可以成功提取纯化出浓度较高的CT3蛋白片段。结论超声破碎法联合应用DTT与PreScission蛋白酶能够提取纯化出高浓度和纯度的CT3蛋白片段,为深入研究CT3的生物学功能奠定了基础。
Objective To investigate the method of extracting and purifying CT3 protein fragment of Ca_V1.2 calcium channel in myocardium. Methods CT3 cDNA was inserted into pG EX-6p-3 plasmid vector and transformed into E. coli BL-21 competent cells. After extensive culture and induction of GST fusion of CT3 by IPTG The protein was expressed and purified by sonication. The GST tag was excised by PreScission protease in the presence of 10 mmol / L DL-dithiothreitol (DTT), and the CT3 protein was identified by gel electrophoresis. The protein purity and Relative molecular mass. Results The results of SDSPAGE showed that the GST-CT3 fusion protein could be purified by sonication and the GST-CT3 protein was digested by PreScission protease alone. The concentration of CT3 protein fragment was lower than that of the original one, while the combination of PreS cission protease and DTT could be successfully used for purification A high concentration of CT3 protein fragments. Conclusion Ultrasound fragmentation combined with DTT and PreScission protease can extract and purify CT3 protein with high concentration and purity, which lays the foundation for further study on the biological function of CT3.