Sandwiched-fusion strategy facilitates recombinant production of small labile proteins
Sandwiched-fusion strategy facilitates recombinant production of small labile proteins
复制标题
夹心融合策略促进小不稳定蛋白的重组生产
DOI：
10.1002/pro.4024
复制
发表时间：
2021-01-29
期刊：
影响因子：
8
通讯作者：
Huang, Chengdong
中科院分区：
文献类型：
--
作者：
Huang, Lin;Qu, Xiaozhan;Huang, Chengdong
在现代结构和功能基因组学中，高效生产大量可溶性的、正确折叠的蛋白质是高需求的。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常常由于快速的蛋白水解降解而不能被回收。在这里，我们表明，基于两个蛋白质标签合并在目标蛋白的氨基和羧基末端的双酶切融合策略，可以用来克服这一障碍。我们已经利用这种策略在大肠杆菌中异源表达的8个小的降解倾向的真核蛋白，其成功的重组生产尚未实现。这些包括七种源自蚊子的肽（MDPS），一类独特的人体代谢调节因子，以及一种不稳定的蚊子转录因子Guy 1。在这里，我们表明，双酶切融合策略，它提供了强大的保护蛋白水解，提供了一种经济的方法，以获得大量的纯5 MDP和转录因子Guy 1，在鲜明的对比，否则不成功的恢复使用传统的氨基融合方法。进一步的生物物理特性和相互作用的研究，通过NMR光谱证实，这种新的方法产生的蛋白质被正确折叠成其生物活性结构。我们预计这种策略可以广泛用于生产其他不稳定的蛋白质系统。
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.