Whi2蛋白的表达与纯化
首发时间:2018-04-20
摘要:目的:研究提高Whi2蛋白产量的方法。方法:将WHI2基因片段插入含GST亲和标签的GH413质粒中,转化至大肠杆菌中。通过在不同温度下,加入不同终浓度IPTG培养不同的时间对GST-Whi2融合蛋白进行诱导表达,筛选出最佳诱导条件。利用Sarkosyl提高可溶性蛋白比例,经谷胱甘肽琼脂糖凝胶珠进行纯化后,通过SDS-PAGE和考马士亮蓝染色检测蛋白的纯度。结果:构建了含GST-WHI2融合基因的质粒,并可在大肠杆菌中用IPTG成功诱导GST-Whi2融合蛋白的表达。通过使用Sarkosyl成功降低了包涵体的生成,提高了融合蛋白的可溶性。最终利用谷胱甘肽琼脂糖凝胶珠纯化GST-Whi2融合蛋白,再用凝血酶酶切得到了高产量和高纯度的Whi2蛋白。纯化后的Whi2蛋白产量达到10 mg/L菌液。结论:成功构建GST-Whi2表达体系,筛选出最佳诱导条件和纯化条件,得到了高纯度的Whi2蛋白。
关键词: GST融合蛋白 Whi2蛋白; 蛋白表达纯化
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Whi2 expression and purification
Abstract:Aim: To find the way to improve the production of Whi2. Methods: Clone WHI2 gene into plasmid GH413 containing GST affinity tap and then transform it into E.coli. GST-Whi2 was induced to express at different temperatures by adding different concentrations of IPTG for different times, and the optimal inducing conditions were selected. Using Sarkosyl to increase the proportion of soluble protein. After purification by glutathione agarose beads, the purity of the protein was detected by SDS-PAGE and Coomassie blue staining. Results: A plasmid containing the GST-WHI2 fusion gene was constructed, and the expression of the GST-Whi2 fusion protein was successfully induced by IPTG in E.coli. The use of Sarkosyl has successfully reduced the generation of inclusion bodies and improved the solubility of the fusion protein. Finally, the GST-Whi2 fusion protein was purified using glutathione agarose beads, and pure Whi2 protein was obtained after digestion by thrombin, with a yield of 10 mg/L culture. Conclusion: The GST-Whi2 expression system was successfully constructed, the best inducing and purification conditions were selected, and high purity Whi2 protein was obtained.
Keywords: GST fusion protein Whi2 protein protein expression and purification
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