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J. Hefei Univ. Tech. (Nat. Sci.)

合肥工业大学学报(自然科学版)

JOURNAL OF HEFEI UNIVERSITY OF TECHNOLOGY (NATURAL SCIENCE)

Competent Authorities: Ministry of Education of the People's Republic of China

Sponsor by: Hefei University of Technology

ISSN 1000-1646, CN 34-1001/N

Optimization for expression, renaturation and purification of PspA inclusion body protein of Cronobacter malonaticus

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丙二酸盐克罗诺杆菌 PspA 包涵体蛋白的表达、复性及纯化方法优化

Journal Information

J. Hefei Univ. Tech. (Nat. Sci.), Jan.2023, Vol.46 No.1: 131-135

DOI: 10.3969/j.issn.1003-5060.2023.01.020

Authors

JIANG Xiuting, LING Na, YE Yingwang

(School of Food and Biological Engineering, Hefei University of Technology, Hefei 230601, China)

Abstract and Keywords

Abstract: To investigate the structure and function of phage shock protein A(PspA) of Cronobacter malonaticus, prokaryotic expression vector carrying PspA was constructed and the protein was expressed in large quantities in the form of inclusion bodies. In order to obtain a high concentration of soluble protein, the urea lysate was used to denature the protein and then the GST-PspA inclusion body protein was renatured to regain its biological activity. The results showed that the dialysis of inclusion body protein by urea gradient dilution at low temperature of 4 ℃ to control the rate of renaturation and prevent the rapid aggregation of protein molecules could efficiently renature and purify GST-PspA fusion protein. The results can provide reference for further exploration of the functions and interactions of the proteins in the Psp system.

Keywords: Cronobacter malonaticus; phage shock protein(Psp) system; vector construction; inclusion body; protein renaturation

作者信息

蒋秀婷，凌娜，叶应旺

（合肥工业大学食品与生物工程学院，安徽合肥230601）

摘要和关键词

摘要: 为研究丙二酸盐克罗诺杆菌(Cronobacter malonaticus)中噬菌体休克蛋白A(phage shock protein A, PspA)的结构与功能,文章构建了PspA融合蛋白表达载体,但重组蛋白以包涵体形式大量表达;为获取较高浓度的可溶性蛋白进行后续探究,使用尿素裂解液使蛋白变性后,经多种方法复性GST-PspA包涵体蛋白,使其重新恢复生物学活性。研究结果表明, $ 4^{\circ} $C低温条件下包涵体蛋白经尿素梯度稀释透析以控制复性速率,防止蛋白分子快速聚集,可以实现高效复性和纯化GST-PspA融合蛋白。为进一步探索Psp系统中各蛋白的功能及相互作用的研究提供一定的参考。

关键词: 丙二酸盐克罗诺杆菌；噬菌体休克蛋白(Psp)系统；载体构建；包涵体；蛋白复性中图分类号：Q936 文献标志码：A 文章编号：1003-5060(2023)01-0131-05

Funding

国家重点研发计划资助项目（2017YFC1601202）；国家自然科学基金资助项目（31671951；31972175）

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