二硫苏糖醇提取纯化Cavl.2钙通道片段CT3蛋白

Purification of CT3 Fragment of Cav1.2 with Dithiothreitol

目的探讨二硫苏糖醇(DTT)有助于Pre Scission蛋白酶切除重组蛋白GST-CT3标签GST的方法。方法在大肠埃希菌BL21中转化入p GEX-6P-3/CT3重组质粒并诱导其表达,用B-PER提取纯化重组蛋白GST-CT3,在10 mmol/L DTT存在的情况下Pre Scission蛋白酶切除GST标签得到高浓度的CT3蛋白,进行SDS-PAGE电泳鉴定CT3蛋白。结果不含DTT情况下,Pre Scission蛋白酶很难切除重组蛋白GST-CT3的标签蛋白GST,而在10 mmol/L DTT存在的情况下,Pre Scission蛋白酶能够切除重组蛋白GST-CT3的标签蛋白GST,得到高浓度的CT3蛋白。结论 10 mmol/L DTT有助于Pre Scission蛋白酶切除重组蛋白GST-CT3的标签蛋白GST,得到高浓度的CT3蛋白。

Objective To explore whether dithiothreitol （DTI＇） is helpful for PreScission Protease to cut off the GST from GST-CT3 protein. Meth- ocls The pGEX-6P-3/CT3 recombinant plasmid was transfected into Escheffchia coli BL21, and the GST-CT3 fusion protein was purified by B- PER method. PreScission Protease was applied with 10 mmol/L DTT to cut off the GST, then the SDS-PAGE was performed for identification of the CT3 protein. Results Without DTF, it was very difficult for PreScission Protease to cut off the GST from GST-CT3 protein. However, in the pres- ence of 10 mmol/L DTT, PreScission Protease could cut off the GST easily as identified by SDS-PAGE. Conclusion 10 mmol/L DTT can help Pre- Scission Protease to cut GST from GST-CT3 protein, so as to achieve high concentration of CT3.