Feng Yufei, Xu Qingyuan, Yang Tao, Sun Encheng, Li Junping, Shi Dongfang, Wu Donglai
Department of Basic Veterinary Medicine, College of Veterinary Medicine, Northeast Agricultural University, 59 Mucai Street, Xiangfang District, Harbin 150030, Heilongjiang Province, PR China; The Key Laboratory of Veterinary Public Health, Ministry of Agriculture, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Maduan Street, Nangang District, Harbin 150001, Heilongjiang Province, PR China.
The Key Laboratory of Veterinary Public Health, Ministry of Agriculture, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Maduan Street, Nangang District, Harbin 150001, Heilongjiang Province, PR China.
Protein Expr Purif. 2014 Jul;99:64-9. doi: 10.1016/j.pep.2014.04.001. Epub 2014 Apr 13.
Many approaches for generating large quantities of recombinant protein in Escherichia coli fuse the protein of interest to a protein tag to enhance solubility and improve recovery. However, the fusion tags can confound downstream applications, as the fusion partner can alter the structure and biological activity of the recombinant protein and proteolytic removal of the fusion tags can be expensive. Here we describe a new system for production of native proteins in E. coli that allows for removal of the fusion tag via intracellular self-cleavage by the human rhinovirus 3C (HRV3C) protease. This system allows for parallel cloning of target protein coding sequences into six different expression vectors, each with a different fusion partner tag to enhance solubility during induction. Temperature-regulated expression of the HRV3C protease allows for intracellular removal of the fusion tag following induction, and the liberated recombinant protein can be purified by affinity chromatography by virtue of a short six-histidine tag. This system will be an attractive approach for the expression and purification of recombinant proteins free of solubility-enhancing fusion tags, and should be amenable to high-throughput applications.
在大肠杆菌中大量生成重组蛋白的许多方法是将目标蛋白与蛋白质标签融合，以提高溶解度并改善回收率。然而，融合标签可能会干扰下游应用，因为融合伴侣可能会改变重组蛋白的结构和生物学活性，且通过蛋白水解去除融合标签可能成本高昂。在此，我们描述了一种在大肠杆菌中生产天然蛋白的新系统，该系统允许通过人鼻病毒3C（HRV3C）蛋白酶进行细胞内自我切割来去除融合标签。该系统允许将目标蛋白编码序列平行克隆到六个不同的表达载体中，每个载体都带有不同的融合伴侣标签，以在诱导过程中提高溶解度。HRV3C蛋白酶的温度调节表达允许在诱导后细胞内去除融合标签，并且释放的重组蛋白可凭借短的六组氨酸标签通过亲和层析进行纯化。该系统将是一种用于表达和纯化不含溶解度增强融合标签的重组蛋白的有吸引力的方法，并且应该适用于高通量应用。
