摘要
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 explore the purification method of the CT3 protein fragment of cardiac Cav1.2 calcium channel. Methods Escherichia coli BL-21 was transformed with the plasmid of pGEX-6p-3/CT3. The CT3-GST fusion protein was mass cultured and induced by isopropyl-β-D-thiogalactoside （IPTG）. Then, the GST-CT3 fusion protein was purified by ultrasonic crushing method. In the presence of 10 mmol/L DTT, PreSeission Protease was applied to cut off the GST, then the SDS-PAGE was used to identify CT3 protein fragment and detect its purity and relative molecular weight. Results The SDS-PAGE results showed that the ultrasonic crushing method can gain and purify GST-CT3 fusion protein, and the concentration of the CT3 protein segment is low by PreScission enzyme alone. But the CT3 protein segment have a high concentration by PreScission enzyme and DTT. Conclusion The simple and easy ultrasonic crushing method and DTT can gain and purify the CT3 protein fragment of the high concentration and purity, and lays a foundation for further research of biological function of CT3.
出处
《解剖科学进展》
2016年第6期602-605,共4页
Progress of Anatomical Sciences
基金
国家自然科学基金(31400981
31471091)
大学生创新计划项目(2015069
201510159068
201510159047
2015048)
关键词
DTT
CT3
融合蛋白
提取
纯化
超声破碎法
DTT
CT3
fusion protein
extraction
purification
ultrasonic crushing method
