Ministry of Education Key Laboratory for Membrane-less Organelles and Cellular Dynamics, Hefei National Laboratory for Physical Sciences at the Microscale, School of Life Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, China.
Protein Sci. 2021 Mar;30(3):650-662. doi: 10.1002/pro.4024. Epub 2021 Jan 29.
Efficient production of large quantities of soluble, properly folded proteins is of high demand in modern structural and functional genomics. Despite much advancement toward improving recombinant protein expression, many eukaryotic proteins especially small peptides often fail to be recovered due to rapid proteolytic degradation. Here we show that the sandwiched-fusion strategy, which is based on two protein tags incorporated both at the amino- and carboxyl-terminus of target protein, could be employed to overcome this obstacle. We have exploited this strategy on heterologous expression in Escherichia coli of eight small degradation-prone eukaryotic proteins, whose successful recombinant productions have yet to be achieved. These include seven mitochondria-derived peptides (MDPS), a class of unique metabolic regulators of human body, and a labile mosquito transcription factor, Guy1. We show here that the sandwiched-fusion strategy, which provides robust protection against proteolysis, affords an economical method to obtain large quantities of pure five MDPs and the transcription factor Guy1, in sharp contrast to otherwise unsuccessful recovery using the traditional amino-fusion method. Further biophysical characterization and interaction studies by NMR spectroscopy confirmed that the proteins produced by this novel approach are properly folded into their biologically active structures. We anticipate this strategy could be widely utilized in production of other labile protein systems.
高效生产大量可溶性、正确折叠的蛋白质是现代结构和功能基因组学的迫切需求。尽管在提高重组蛋白表达方面取得了很大进展，但许多真核蛋白，特别是小肽，由于快速的蛋白水解降解，往往无法回收。在这里，我们展示了基于靶蛋白氨基和羧基末端同时整合两个蛋白标签的夹心融合策略，可以克服这一障碍。我们已经在大肠杆菌中外源表达了 8 种易降解的真核小蛋白，这些蛋白的重组表达尚未成功。其中包括 7 种线粒体衍生肽（MDPS），一类独特的人体代谢调节剂，以及不稳定的蚊子转录因子 Guy1。我们在这里展示，夹心融合策略提供了对蛋白水解的强大保护，提供了一种经济的方法来获得大量的纯 5 个 MDPS 和转录因子 Guy1，与传统的氨基融合方法不成功的回收形成鲜明对比。进一步的生物物理特性分析和通过 NMR 光谱进行的相互作用研究证实，通过这种新方法产生的蛋白质正确折叠成具有生物活性的结构。我们预计这种策略可以广泛应用于其他不稳定蛋白系统的生产。
