Strain engineering for improved expression of recombinant proteins in bacteria

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Abstract

Protein expression in Escherichia coli represents the most facile approach for the preparation of non-glycosylated proteins for analytical and preparative purposes. So far, the optimization of recombinant expression has largely remained a matter of trial and error and has relied upon varying parameters, such as expression vector, media composition, growth temperature and chaperone co-expression. Recently several new approaches for the genome-scale engineering of E. coli to enhance recombinant protein expression have been developed. These methodologies now enable the generation of optimized E. coli expression strains in a manner analogous to metabolic engineering for the synthesis of low-molecular-weight compounds. In this review, we provide an overview of strain engineering approaches useful for enhancing the expression of hard-to-produce proteins, including heterologous membrane proteins.

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Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels.

B Miroux, J. Walker (1996)

We have investigated the over-production of seven membrane proteins in an Escherichia coli-bacteriophage T7 RNA polymerase expression system. In all seven cases, when expression of the target membrane protein was induced, most of the BL21(DE3) host cells died. Similar effects were also observed with expression vectors for ten globular proteins. Therefore, protein over-production in this expression system is either limited or prevented by bacterial cell death. From the few survivors of BL21(DE3) expressing the oxoglutarate-malate carrier protein from mitochondrial membranes, a mutant host C41(DE3) was selected that grew to high saturation cell density, and produced the protein as inclusion bodies at an elevated level without toxic effect. Some proteins that were expressed poorly in BL21(DE3), and others where the toxicity of the expression plasmids prevented transformation into this host, were also over-produced successfully in C41(DE3). The examples include globular proteins as well as membrane proteins, and therefore, strain C41(DE3) is generally superior to BL21(DE3) as a host for protein over-expression. However, the toxicity of over-expression of some of the membrane proteins persisted partially in strain C41(DE3). Therefore, a double mutant host C43(DE3) was selected from C41(DE3) cells containing the expression plasmid for subunit b of bacterial F-ATPase. In strain C43(DE3), both subunits b and c of the F-ATPase, an alanine-H(+) symporter, and the ADP/ATP and the phosphate carriers from mitochondria were all over-produced. The transcription of the gene for the OGCP and subunit b was lower in C41(DE3) and C43(DE3), respectively, than in BL21(DE3). In C43(DE3), the onset of transcription of the gene for subunit b was delayed after induction, and the over-produced protein was incorporated into the membrane. The procedure used for selection of C41(DE3) and C43(DE3) could be employed to tailor expression hosts in order to overcome other toxic effects associated with over-expression.

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Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein.

Stéphanie Cabantous, Thomas Terwilliger, Geoffrey S. Waldo (2005)

Existing protein tagging and detection methods are powerful but have drawbacks. Split protein tags can perturb protein solubility or may not work in living cells. Green fluorescent protein (GFP) fusions can misfold or exhibit altered processing. Fluorogenic biarsenical FLaSH or ReASH substrates overcome many of these limitations but require a polycysteine tag motif, a reducing environment and cell transfection or permeabilization. An ideal protein tag would be genetically encoded, would work both in vivo and in vitro, would provide a sensitive analytical signal and would not require external chemical reagents or substrates. One way to accomplish this might be with a split GFP, but the GFP fragments reported thus far are large and fold poorly, require chemical ligation or fused interacting partners to force their association, or require coexpression or co-refolding to produce detectable folded and fluorescent GFP. We have engineered soluble, self-associating fragments of GFP that can be used to tag and detect either soluble or insoluble proteins in living cells or cell lysates. The split GFP system is simple and does not change fusion protein solubility.

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Recombinant protein folding and misfolding in Escherichia coli.

François Baneyx, Mirna Mujacic (2004)

The past 20 years have seen enormous progress in the understanding of the mechanisms used by the enteric bacterium Escherichia coli to promote protein folding, support protein translocation and handle protein misfolding. Insights from these studies have been exploited to tackle the problems of inclusion body formation, proteolytic degradation and disulfide bond generation that have long impeded the production of complex heterologous proteins in a properly folded and biologically active form. The application of this information to industrial processes, together with emerging strategies for creating designer folding modulators and performing glycosylation all but guarantee that E. coli will remain an important host for the production of both commodity and high value added proteins.

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Author and article information

Journal

Journal ID (nlm-ta): Microb Cell Fact

Title: Microbial Cell Factories

Publisher: BioMed Central

ISSN (Electronic): 1475-2859

Publication date Collection: 2011

Publication date (Electronic): 14 May 2011

Volume: 10

Page: 32

Affiliations

[1 ]Department of Chemical Engineering, The University of Texas at Austin, Austin, Texas 78712, USA

[2 ]Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, Texas 78712, USA

[3 ]Department of Biomedical Engineering, The University of Texas at Austin, Austin, Texas 78712, USA

[4 ]Section of Molecular Genetics and Microbiology, The University of Texas at Austin, Austin, Texas 78712, USA

[5 ]Insitute of Biological Research and Biotechnology, National Hellenic Research Foundation, 48 Vassileos Constantinou Ave., 11635 Athens, Greece

[6 ]Asubio Pharma CO., LTD. 6-4-3, Minatojima-Minamimachi Chuo-ku, Kobe 650-0047, Japan

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Publisher ID: 1475-2859-10-32

DOI: 10.1186/1475-2859-10-32

PMC ID: 3120638

PubMed ID: 21569582

SO-VID: 7eb50e4c-2a7a-40d5-a1fe-1e334b254965

License:

This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Strain engineering for improved expression of recombinant proteins in bacteria

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Most cited references 79

Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels.

Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein.

Recombinant protein folding and misfolding in Escherichia coli.

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