高通量结核分枝杆菌蛋白可溶表达筛选平台的构建与应用

Construction and application of a high throughput screening platform for soluble expression of Mycobacterium tuberculosis proteins

目的为了改变结核分枝杆菌蛋白质的结构与功能研究由于受结核分枝杆菌蛋白难以可溶性表达和纯化的影响而进展缓慢的问题。方法将多个能促进可溶性表达的蛋白标签与高通量的Gateway克隆技术相结合，构建结核分枝杆菌蛋白可溶性表达快速通量的筛选平台。挑选46个不同分子量且已知以包涵体形式表达或者不表达的结核分枝杆菌蛋白的基因，分别克隆到带有N-末端的氮源利用物质A（N utilization substance A，NusA）、小分子泛素样修饰蛋白（small ubiquitin-like modifier-1，SUMO）、麦芽糖结合蛋白（maltose-binding protein，MBP）、硫氧还蛋白（thioredoxin A，TrxA）、酰基载体蛋白（acyl carrier protein，ACP）五种蛋白标签基因的载体上，进行融合表达，分析比较各标签促进可溶性表达的效果。结果融合NusA标签后，19个蛋白以可溶性形式表达，占总样本的41％（19／46）；同时融合SUMO、MBP、TrxA、ACP标签后，可溶性表达的蛋白也分别达到总样本数的22％（10／46）、26％（12／46）、26％（12／46）、22％（10／46）。结论结核分枝杆菌蛋白可溶性表达快速通量筛选平台的建立，实现了结核分枝杆菌重组蛋白可溶性表达的快速通量筛选，为更进一步研究结核分枝杆菌蛋白、特别是重要药物靶标蛋白的结构与功能创造了条件。

Objective To solve the problem that the progress in the study of Mycobacterium tuberculosis protein structure and function is slow as M. tuberculosis proteins are difficult to express and purify in soluble form. Methods We developed a high-throughput screening platform for the soluble expression of M. tuberculosis proteins in Escherichia coli by combining high-throughput Gateway cloning with the use of multiple fusion tags. We randomly selected 46 genes for M. tuberculosis proteins of different molecular weights which have been difficult to purify as they are either not expressed or are expressed in inclusion bodies and fused them to the C-terminals of the N utilization substance A （NusA）, small ubiquitin-like modifier-1 （SUMO）, maltose-binding protein （MBP）, thioredoxin A （TrxA）, and acyl carrier protein （ACP） tags. These constructs were then expressed in E. coli and evaluated for expression and solubility. Results As expected, the fusion tags varied in their ability to produce soluble M. tuberculosis proteins. NusA fusion enhanced expression and solubility of recombinant proteins most dramatically; 19 proteins （41%,19/46） fused with the NusA tag were expressed in soluble form, while 22% （10/46）, 26% （12/46）, 26%（12/46） and 22%（10/46）of proteins fused with the SUMO, MBP, TrxA, and ACP tags, respectively, were solubly expressed. Conclusion The development of high throughput screening platform for the soluble expression of M. tuberculosis proteins provides a screening platform for the rapid high-throughput soluble expression of M. tuberculosis recombinant proteins and will facilitate the structural and functional studies of M. tuberculosis proteins, especially the proteins targeted by the important drugs.