Development of a Protein-assisted Protein Overexpression System for Membrane Proteins

膜蛋白質占各種生物細胞內所有蛋白質的20~30%，且在生理功能上扮演重要的角色。要研究膜蛋白質的功能及機制，需要取得足夠量的蛋白質以進行結晶及 in vitro 分析，而膜蛋白質表現量相當低，這也是目前在 RCSB PDB 蛋白質結構檔案裡僅有約1%為膜蛋白質結構的主要原因。為了提高蛋白質表現量，利用融合蛋白質的方式在大腸桿菌中異源表現是個常用的方法，但是只有少數能同樣運用在膜蛋白質上面。本研究試著發展一套能用來加強或穩定地表現及純化膜蛋白質的系統。此系統採用 Haloarcula marismortui 上發現的光感蛋白質 HmBRI 的突變蛋白質 (命名為 HEBR)。HEBR 在大腸桿菌中具有很大的表現量 (40 mg/L culture)，穩定性相當良好，因此本研究將欲表現之目標膜蛋白與 HEBR 融合，藉由 HEBR 這些特性來幫助目標膜蛋白質表現。此外，HEBR 所帶有的紫色亦可目測及幫助目標膜蛋白的純化。融合策略：HEBR 置於融合蛋白質的 N 端，以發揮其高表現量以及轉運的特性，目標膜蛋白質則位於融合蛋白質的 C 端，並在其前面設計蛋白酶的切位以便截切，由於本研究使用的目標蛋白質皆為 N 端在胞外的膜蛋白質，因此在HEBR的C端額外加上一個穿膜區序列。本研究成功使用 HEBR 融合表現系統輔助表現膜蛋白質，利用功能性測試確認蛋白質摺疊正確。結果顯示此表現系統除了可輔助目標蛋白質表現外，亦可增加目標蛋白質的穩定性。Membrane proteins constitute approximately 20-30% of any prokaryotic or eukaryotic proteome and play important roles in biological systems. A prerequisite to study the functions and mechanisms of membrane proteins is to obtain ample quantity of proteins for in vitro assay and crystallization. Low level of membrane proteins expression in their native host cells or designed expression systems is one of the main reasons that only 1% RCSB PDB entries are membrane proteins structure. Using fusion proteins with E. coli heterologous expression system to increase protein expression level have been adopted before; however, only limited successful fusion membrane protein had been reported. We intend to develop a universal system to assist membrane proteins overexpression and purification. This system utilized a designated mutant based on HmBRI, one of the six new retinal-binding membrane proteins from Haloarcula marismortui. This special designed mutant, HEBR, led to unusual high-level expression in E. coli with a yield as high as 40 mg/L culture, and it was fused to the target proteins as a tag to assist expression. For membrane proteins with the N-terminus starts from periplasmic region, the strategy was designed that the C-terminus of HEBR connects to an additional transmembrane domain from HmHtrI, then it further fuses with the target membrane proteins. Additionally, HEBR has visual color and it is extremely stable; therefore, any protein assisted by HEBR system can be visualized by unaided eyes. Here, we report several membrane proteins successful expressed with our HEBR system. The results showed both increased yields and/or elevated protein stability can be achieved