Optimizing E. coli-Based Membrane Protein Production Using Lemo21(DE3) or pReX and GFP-Fusions

2017 ◽  
pp. 109-126 ◽  
Author(s):  
Grietje Kuipers ◽  
Markus Peschke ◽  
Nurzian Bernsel Ismail ◽  
Anna Hjelm ◽  
Susan Schlegel ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Protein Production ◽  
E Coli

Membrane Protein Production in E. coli Lysates in Presence of Preassembled Nanodiscs

2017 ◽  
pp. 291-312 ◽  
Author(s):  
Ralf-Bernhardt Rues ◽  
Alexander Gräwe ◽  
Erik Henrich ◽  
Frank Bernhard
Keyword(s):  
Membrane Protein ◽  
Protein Production ◽  
E Coli

scholarly journals Isolation and characterization of the E. coli membrane protein production strain Mutant56(DE3)

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Thomas Baumgarten ◽  
Susan Schlegel ◽  
Samuel Wagner ◽  
Mirjam Löw ◽  
Jonas Eriksson ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Protein Production ◽  
E Coli ◽  
Isolation And Characterization ◽  
Production Strain

Optimization of Membrane Protein Production Using Titratable Strains of E. coli

2017 ◽  
pp. 83-107
Author(s):  
Rosa Morra ◽  
Kate Young ◽  
David Casas-Mao ◽  
Neil Dixon ◽  
Louise E. Bird
Keyword(s):  
Membrane Protein ◽  
Protein Production ◽  
E Coli

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.

Optimizing E. coli-Based Membrane Protein Production Using Lemo21(DE3) and GFP-Fusions

2013 ◽  
pp. 381-400 ◽  
Author(s):  
Anna Hjelm ◽  
Susan Schlegel ◽  
Thomas Baumgarten ◽  
Mirjam Klepsch ◽  
David Wickström ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Protein Production ◽  
E Coli

scholarly journals Development and comparison of cell-free protein synthesis systems derived from typical bacterial chassis

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Liyuan Zhang ◽  
Xiaomei Lin ◽  
Ting Wang ◽  
Wei Guo ◽  
Yuan Lu
Keyword(s):  
Protein Synthesis ◽  
Protein Production ◽  
Influential Factors ◽  
Competent Cell ◽  
Free Protein ◽  
Application Range ◽  
E Coli ◽  
Short Time ◽  
Cell Free Protein Synthesis

AbstractCell-free protein synthesis (CFPS) systems have become an ideal choice for pathway prototyping, protein production, and biosensing, due to their high controllability, tolerance, stability, and ability to produce proteins in a short time. At present, the widely used CFPS systems are mainly based on Escherichia coli strain. Bacillus subtilis, Corynebacterium glutamate, and Vibrio natriegens are potential chassis cells for many biotechnological applications with their respective characteristics. Therefore, to expand the platform of the CFPS systems and options for protein production, four prokaryotes, E. coli, B. subtilis, C. glutamate, and V. natriegens were selected as host organisms to construct the CFPS systems and be compared. Moreover, the process parameters of the CFPS system were optimized, including the codon usage, plasmid synthesis competent cell selection, plasmid concentration, ribosomal binding site (RBS), and CFPS system reagent components. By optimizing and comparing the main influencing factors of different CFPS systems, the systems can be optimized directly for the most influential factors to further improve the protein yield of the systems. In addition, to demonstrate the applicability of the CFPS systems, it was proved that the four CFPS systems all had the potential to produce therapeutic proteins, and they could produce the receptor-binding domain (RBD) protein of SARS-CoV-2 with functional activity. They not only could expand the potential options for in vitro protein production, but also could increase the application range of the system by expanding the cell-free protein synthesis platform.

scholarly journals Random and combinatorial mutagenesis for improved total production of secretory target protein in Escherichia coli

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
David Gonzalez-Perez ◽  
James Ratcliffe ◽  
Shu Khan Tan ◽  
Mary Chen May Wong ◽  
Yi Pei Yee ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein Secretion ◽  
Protein Production ◽  
Secretory Protein ◽  
Target Protein ◽  
Carrier Protein ◽  
Signal Peptides ◽  
Carrier Proteins ◽  
E Coli ◽  
Combinatorial Mutagenesis

AbstractSignal peptides and secretory carrier proteins are commonly used to secrete heterologous recombinant protein in Gram-negative bacteria. The Escherichia coli osmotically-inducible protein Y (OsmY) is a carrier protein that secretes a target protein extracellularly, and we have previously applied it in the Bacterial Extracellular Protein Secretion System (BENNY) to accelerate directed evolution. In this study, we reported the first application of random and combinatorial mutagenesis on a carrier protein to enhance total secretory target protein production. After one round of random mutagenesis followed by combining the mutations found, OsmY(M3) (L6P, V43A, S154R, V191E) was identified as the best carrier protein. OsmY(M3) produced 3.1 ± 0.3 fold and 2.9 ± 0.8 fold more secretory Tfu0937 β-glucosidase than its wildtype counterpart in E. coli strains BL21(DE3) and C41(DE3), respectively. OsmY(M3) also produced more secretory Tfu0937 at different cultivation temperatures (37 °C, 30 °C and 25 °C) compared to the wildtype. Subcellular fractionation of the expressed protein confirmed the essential role of OsmY in protein secretion. Up to 80.8 ± 12.2% of total soluble protein was secreted after 15 h of cultivation. When fused to a red fluorescent protein or a lipase from Bacillus subtillis, OsmY(M3) also produced more secretory protein compared to the wildtype. In this study, OsmY(M3) variant improved the extracellular production of three proteins originating from diverse organisms and with diverse properties, clearly demonstrating its wide-ranging applications. The use of random and combinatorial mutagenesis on the carrier protein demonstrated in this work can also be further extended to evolve other signal peptides or carrier proteins for secretory protein production in E. coli.

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