| 陈 丽,吴炳男,黄 晨,肖 树,陆昌瑞,何创龙,陈 婷.RNA编辑蛋白OTP82的表达与纯化[J].,2022,(15):2807-2812 |
| RNA编辑蛋白OTP82的表达与纯化 |
| Expression and Purification of RNA Editing Protein OTP82 |
| 投稿时间：2022-01-28 修订日期：2022-02-23 |
| DOI：10.13241/j.cnki.pmb.2022.15.002 |
| 中文关键词: PPR蛋白 RNA 编辑 OTP82 蛋白纯化 |
| 英文关键词: PPR protein RNA editing OTP82 Protein purification |
| 基金项目:上海市科学技术委员会国际合作计划项目；（19410711000）；上海市科学技术委员会自然科学基金项目（19ZR1471100） |
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| 中文摘要: |
| 摘要 目的：利用原核系统表达RNA编辑蛋白OTP82，通过蛋白变复性的方法得到全长蛋白，并优化实验条件提高OTP82的收率。方法：利用原核表达系统表达OTP82全长融合蛋白（HSO），表达后的菌体用高压细胞破碎仪匀浆后收集包涵体并用包涵体洗涤液重复洗涤两遍，然后用含有8 M尿素的变性缓冲液将包涵体搅拌溶解得到蛋白原始液。将蛋白原始液复性后采用镍柱亲和纯化，通过SDS-PAGE和Western-blot检测等方法对HSO的变复性结果进行筛选。结果：通过对复性缓冲液中盐浓度、谷胱甘肽的浓度和比例以及小分子添加剂的探索，得到了OTP82融合蛋白适合的变复性条件（pH 8.5 100 mM Tris，400 mM NaCl，200 mM 精氨酸，5 mM GSH，0.5 mM GSSG，6 mM β-环糊精，2 mM EDTA，1 mM PMSF）复性率达到2.73%（获得蛋白0.42 mg/L）。结论：通过变复性的方式能够在体外得到OTP82的粗蛋白，为揭示RNA编辑蛋白作用机制提供基础实验依据，为后续利用PPR蛋白进行工程蛋白设计奠定了基础。 |
| 英文摘要: |
| ABSTRACT Objective: Express the RNA editing protein OTP82 by the prokaryotic system to, obtain the full-length protein by protein denaturation and renaturation, and optimize the experimental conditions to increase the yield of OTP82. Methods: The OTP82 full-length fusion protein (HSO) was expressed by a prokaryotic expression system. The expressed bacterial cells were crushed under high pressure, then collected the inclusion body. Washed the collected inclusion body twice with washing solution, and then dissolved it with the denaturation buffer containing 8 M urea to obtain the original protein solution. Purified the protein after refolding with nickel columns. The results of denaturation and renaturation of OTP82 full-length fusion protein (HSO) were screened by SDS-PAGE and Western-blot detection. Results: Through the exploration of the concentration of sodium chloride, the concentration and ratio of glutathione, and small molecule additives in the refolding buffer, obtained suitable denaturation conditions for HSO protein (pH 8.5 100 mM Tris, 400 mM NaCl, 200 mM Arginine, 5 mM GSH, 0.5 mM GSSG, 6 mM β-Cyclodextrin, 2 mM EDTA, 1 mM PMSF) and reproduction rate reached 2.73% (0.42 mg/L). Conclusion: The crude protein of OTP82 can be obtained in vitro by denaturation, which provides a basic experimental basis for revealing the mechanism of action of RNA editing protein and lays the foundation for subsequent engineering protein design using PPR protein. |
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