Development of a Protein-assisted Protein Overexpression System for Membrane Proteins
Date Issued
2011
Date
2011
Author(s)
Hsieh, Shiang-Yuan
Abstract
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.
Subjects
membrane protein
membrane protein expression system
Type
thesis
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