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Title: Improving membrane protein expression and function using genomic edits

Expression of membrane proteins often leads to growth inhibition and perturbs central metabolism and this burden varies with the protein being overexpressed. There are also known strain backgrounds that allow greater expression of membrane proteins but that differ in efficacy across proteins. Here, we hypothesized that for any membrane protein, it may be possible to identify a modified strain background where its expression can be accommodated with less burden. To directly test this hypothesis, we used a bar-coded transposon insertion library in tandem with cell sorting to assess genome-wide impact of gene deletions on membrane protein expression. The expression of five membrane proteins (CyoB, CydB, MdlB, YidC, and LepI) and one soluble protein (GST), each fused to GFP, was examined. We identified Escherichia coli mutants that demonstrated increased membrane protein expression relative to that in wild type. For two of the proteins (CyoB and CydB), we conducted functional assays to confirm that the increase in protein expression also led to phenotypic improvement in function. This study represents a systematic approach to broadly identify genetic loci that can be used to improve membrane protein expression, and our method can be used to improve expression of any protein that poses a cellularmore » burden.« less
Authors:
 [1] ;  [1] ;  [1] ;  [1] ;  [1]
  1. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division
Publication Date:
Grant/Contract Number:
AC02-05CH11231
Type:
Accepted Manuscript
Journal Name:
Scientific Reports
Additional Journal Information:
Journal Volume: 7; Journal Issue: 1; Journal ID: ISSN 2045-2322
Publisher:
Nature Publishing Group
Research Org:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; Applied microbiology; Molecular biology
OSTI Identifier:
1419447

Jensen, Heather M., Eng, Thomas, Chubukov, Victor, Herbert, Robin A., and Mukhopadhyay, Aindrila. Improving membrane protein expression and function using genomic edits. United States: N. p., Web. doi:10.1038/s41598-017-12901-7.
Jensen, Heather M., Eng, Thomas, Chubukov, Victor, Herbert, Robin A., & Mukhopadhyay, Aindrila. Improving membrane protein expression and function using genomic edits. United States. doi:10.1038/s41598-017-12901-7.
Jensen, Heather M., Eng, Thomas, Chubukov, Victor, Herbert, Robin A., and Mukhopadhyay, Aindrila. 2017. "Improving membrane protein expression and function using genomic edits". United States. doi:10.1038/s41598-017-12901-7. https://www.osti.gov/servlets/purl/1419447.
@article{osti_1419447,
title = {Improving membrane protein expression and function using genomic edits},
author = {Jensen, Heather M. and Eng, Thomas and Chubukov, Victor and Herbert, Robin A. and Mukhopadhyay, Aindrila},
abstractNote = {Expression of membrane proteins often leads to growth inhibition and perturbs central metabolism and this burden varies with the protein being overexpressed. There are also known strain backgrounds that allow greater expression of membrane proteins but that differ in efficacy across proteins. Here, we hypothesized that for any membrane protein, it may be possible to identify a modified strain background where its expression can be accommodated with less burden. To directly test this hypothesis, we used a bar-coded transposon insertion library in tandem with cell sorting to assess genome-wide impact of gene deletions on membrane protein expression. The expression of five membrane proteins (CyoB, CydB, MdlB, YidC, and LepI) and one soluble protein (GST), each fused to GFP, was examined. We identified Escherichia coli mutants that demonstrated increased membrane protein expression relative to that in wild type. For two of the proteins (CyoB and CydB), we conducted functional assays to confirm that the increase in protein expression also led to phenotypic improvement in function. This study represents a systematic approach to broadly identify genetic loci that can be used to improve membrane protein expression, and our method can be used to improve expression of any protein that poses a cellular burden.},
doi = {10.1038/s41598-017-12901-7},
journal = {Scientific Reports},
number = 1,
volume = 7,
place = {United States},
year = {2017},
month = {10}
}

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