A novel yeast expression system for the overproduction of quality-controlled membrane proteins

被引:39
作者
Griffith, DA
Delipala, C
Leadsham, J
Jarvis, SM
Oesterhelt, D
机构
[1] Max Planck Inst Biochem, Dept Membrane Biochem, D-82152 Martinsried, Germany
[2] Univ Westminster, Sch Biosci, London W1W 6UW, England
关键词
membrane protein; protein overexpression; protein folding; unfolded protein response; Saccharomyces cerevisiae;
D O I
10.1016/S0014-5793(03)00952-9
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Saturation of the cell's protein folding capacity and accumulation of inactive incompletely folded protein often accompanying the overexpression of membrane proteins (MPs) presents an obstacle to their efficient purification in a functional form for structural studies. We present a novel strategy for optimization of functional NIP expression in Saccharomyces cerevisiae. This approach exploits the unfolded protein response (UPR) pathway, a stress signaling mechanism that senses the accumulation of unfolded proteins in the endoplasmic reticulum. We demonstrate that a high level of UPR induction upon expression of a NIP reflects impaired functional expression of that protein. Tuning the expression level of the protein so as to avoid or minimize UPR induction results in its increased functional expression. UPR status can therefore serve as a proxy variable for the extent of impaired expression of a MP that may even be applicable in the absence of knowledge of the protein's biological function. (C) 2003 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
引用
收藏
页码:45 / 50
页数:6
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