scholarly journals The E. coli single protein production system for production and structural analysis of membrane proteins

2009 ◽  
Vol 11 (1) ◽  
pp. 81-84 ◽  
Author(s):  
Lili Mao ◽  
S. Thangminlal Vaiphei ◽  
Tsutomu Shimazu ◽  
William M. Schneider ◽  
Yuefeng Tang ◽  
...  
Keyword(s):  
Structural Analysis ◽  
Membrane Proteins ◽  
Production System ◽  
Protein Production ◽  
E Coli ◽  
Single Protein

scholarly journals The Use of the Condensed Single Protein Production System for Isotope-Labeled Outer Membrane Proteins, OmpA and OmpX in E. coli

2010 ◽  
Vol 47 (3) ◽  
pp. 205-210 ◽  
Author(s):  
S. Thangminlal Vaiphei ◽  
Yuefeng Tang ◽  
Gaetano T. Montelione ◽  
Masayori Inouye
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Production System ◽  
Protein Production ◽  
Outer Membrane Proteins ◽  
E Coli ◽  
Single Protein

scholarly journals Secretion of human superoxide dismutase in Escherichia coli using the condensed single-protein-production system

2010 ◽  
Vol 19 (12) ◽  
pp. 2330-2335 ◽  
Author(s):  
Lili Mao ◽  
Peter B. Stathopulos ◽  
Mitsuhiko Ikura ◽  
Masayori Inouye
Keyword(s):  
Escherichia Coli ◽  
Superoxide Dismutase ◽  
Production System ◽  
Protein Production ◽  
Single Protein

scholarly journals High Throughput Expression Screening of Arabinofuranosyltransferases from Mycobacteria

Processes ◽  
2021 ◽  
Vol 9 (4) ◽  
pp. 629
Author(s):  
José Rodrigues ◽  
Vanessa T. Almeida ◽  
Ana L. Rosário ◽  
Yong Zi Tan ◽  
Brian Kloss ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Structural Biology ◽  
High Throughput ◽  
Drug Targets ◽  
Large Scale ◽  
Protein Production ◽  
Expression Screening ◽  
E Coli ◽  
Metal Affinity ◽  
Mycobacterial Cell Wall

Studies on membrane proteins can help to develop new drug targets and treatments for a variety of diseases. However, membrane proteins continue to be among the most challenging targets in structural biology. This uphill endeavor can be even harder for membrane proteins from Mycobacterium species, which are notoriously difficult to express in heterologous systems. Arabinofuranosyltransferases are involved in mycobacterial cell wall synthesis and thus potential targets for antituberculosis drugs. A set of 96 mycobacterial genes coding for Arabinofuranosyltransferases was selected, of which 17 were successfully expressed in E. coli and purified by metal-affinity chromatography. We herein present an efficient high-throughput strategy to screen in microplates a large number of targets from Mycobacteria and select the best conditions for large-scale protein production to pursue functional and structural studies. This methodology can be applied to other targets, is cost and time effective and can be implemented in common laboratories.

scholarly journals Simple Genetic Selection Protocol for Isolation of Overexpressed Genes That Enhance Accumulation of Membrane-Integrated Human G Protein-Coupled Receptors in Escherichia coli

2010 ◽  
Vol 76 (17) ◽  
pp. 5852-5859 ◽  
Author(s):  
Georgios Skretas ◽  
George Georgiou
Keyword(s):  
Escherichia Coli ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
G Protein ◽  
Protein Production ◽  
Genetic Selection ◽  
G Protein Coupled Receptors ◽  
Efficient Production ◽  
E Coli ◽  
G Protein Coupled

ABSTRACT The efficient production of membrane proteins in bacteria remains a major challenge. In this work, we sought to identify overexpressed genes that enhance the yields of recombinant membrane proteins in Escherichia coli. We developed a genetic selection system for bacterial membrane protein production, consisting of membrane protein fusions with the enzyme β-lactamase and facile selection of high-production strains on ampicillin-containing media. This system was used to screen the ASKA library, an ordered library of plasmids encoding all the known E. coli open reading frames (ORFs), and several clones with the ability to accumulate enhanced amounts of recombinant membrane proteins were selected. Notably, coexpression of ybaB, a gene encoding a putative DNA-binding protein of unknown function, was found to enhance the accumulation of a variety of membrane-integrated human G protein-coupled receptors and other integral membrane proteins in E. coli by up to 10-fold. The results of this study highlight the power of genetic approaches for identifying factors that impact membrane protein biogenesis and for generating engineered microbial hosts for membrane protein production.

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