Abstract
Eukaryotic integral membrane proteins (IMPs) are difficult to study due to low functional expression levels. To investigate factors for efficient biogenesis of eukaryotic IMPs in the prokaryotic model organism Escherichia coli, important, e.g., for isotope-labeling for NMR, we selected for E. coli cells expressing high levels of functional G protein-coupled receptors (GPCRs) by FACS. Utilizing an E. coli strain library with all non-essential genes systematically deleted, we unexpectedly discovered upon whole-genome sequencing that the improved phenotype was not conferred by the deleted genes but by various subtle alterations in the “housekeeping” sigma 70 factor (RpoD). When analyzing effects of the rpoD mutations at the transcriptome level we found that toxic effects incurred on wild-type E. coli during receptor expression were diminished by two independent and synergistic effects: a slower but longer-lasting GPCR biosynthesis and an optimized transcriptional pattern, augmenting growth and expression at low temperature, setting the basis for further bacterial strain engineering.
Highlights
Integral membrane proteins (IMPs) have many vital biological functions, constituting approximately one third of all proteins in humans as well as being the targets of nearly 60% of all FDA-approved drugs[1,2]
In order to identify E. coli genes that could affect the heterologous production of G protein-coupled receptors (GPCRs), we used a selection strategy based on fluorescence-activated cell sorting (FACS) developed in our laboratory that allows the isolation of cells showing increased expression levels of functional receptors that bind to a fluorescent ligand[10,11,12]
While we initially started off from a single-gene deletion strategy in searching for strains with improved functional GPCR expression properties, we found no single gene with these properties
Summary
Integral membrane proteins (IMPs) have many vital biological functions, constituting approximately one third of all proteins in humans as well as being the targets of nearly 60% of all FDA-approved drugs[1,2]. Despite their importance, structural and functional information for IMPs is still limited. While bacteria are able to produce some of their endogenous membrane proteins in high abundance, many IMPs, especially those of eukaryotic origin, are very toxic for the bacterial cell when overexpressed. The titration of the Sec translocon, the limited availability of other endogenous factors assisting in the biogenesis of membrane proteins, or differences in membrane bilayer properties and membrane space can all affect insertion, folding and functioning of heterologous IMPs15,16
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
