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      Recombinant protein expression in Escherichia coli: advances and challenges

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          Abstract

          Escherichia coli is one of the organisms of choice for the production of recombinant proteins. Its use as a cell factory is well-established and it has become the most popular expression platform. For this reason, there are many molecular tools and protocols at hand for the high-level production of heterologous proteins, such as a vast catalog of expression plasmids, a great number of engineered strains and many cultivation strategies. We review the different approaches for the synthesis of recombinant proteins in E. coli and discuss recent progress in this ever-growing field.

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          Most cited references249

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          Protein production by auto-induction in high density shaking cultures.

          Inducible expression systems in which T7 RNA polymerase transcribes coding sequences cloned under control of a T7lac promoter efficiently produce a wide variety of proteins in Escherichia coli. Investigation of factors that affect stability, growth, and induction of T7 expression strains in shaking vessels led to the recognition that sporadic, unintended induction of expression in complex media, previously reported by others, is almost certainly caused by small amounts of lactose. Glucose prevents induction by lactose by well-studied mechanisms. Amino acids also inhibit induction by lactose during log-phase growth, and high rates of aeration inhibit induction at low lactose concentrations. These observations, and metabolic balancing of pH, allowed development of reliable non-inducing and auto-inducing media in which batch cultures grow to high densities. Expression strains grown to saturation in non-inducing media retain plasmid and remain fully viable for weeks in the refrigerator, making it easy to prepare many freezer stocks in parallel and use working stocks for an extended period. Auto-induction allows efficient screening of many clones in parallel for expression and solubility, as cultures have only to be inoculated and grown to saturation, and yields of target protein are typically several-fold higher than obtained by conventional IPTG induction. Auto-inducing media have been developed for labeling proteins with selenomethionine, 15N or 13C, and for production of target proteins by arabinose induction of T7 RNA polymerase from the pBAD promoter in BL21-AI. Selenomethionine labeling was equally efficient in the commonly used methionine auxotroph B834(DE3) (found to be metE) or the prototroph BL21(DE3).
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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.

            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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              Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter.

              We have constructed a series of plasmid vectors (pBAD vectors) containing the PBAD promoter of the araBAD (arabinose) operon and the gene encoding the positive and negative regulator of this promoter, araC. Using the phoA gene and phoA fusions to monitor expression in these vectors, we show that the ratio of induction/repression can be 1,200-fold, compared with 50-fold for PTAC-based vectors. phoA expression can be modulated over a wide range of inducer (arabinose) concentrations and reduced to extremely low levels by the presence of glucose, which represses expression. Also, the kinetics of induction and repression are very rapid and significantly affected by the ara allele in the host strain. Thus, the use of this system which can be efficiently and rapidly turned on and off allows the study of important aspects of bacterial physiology in a very simple manner and without changes of temperature. We have exploited the tight regulation of the PBAD promoter to study the phenotypes of null mutations of essential genes and explored the use of pBAD vectors as an expression system.
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                Author and article information

                Contributors
                Journal
                Front Microbiol
                Front Microbiol
                Front. Microbiol.
                Frontiers in Microbiology
                Frontiers Media S.A.
                1664-302X
                17 April 2014
                2014
                : 5
                : 172
                Affiliations
                [1] 1Instituto de Biología Molecular y Celular de Rosario, Consejo Nacional de Investigaciones Científicas y Técnicas Rosario, Argentina
                [2] 2Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario Rosario, Argentina
                Author notes

                Edited by: Peter Neubauer, Technische Universität Berlin, Germany

                Reviewed by: Jose M. Bruno-Barcena, North Carolina State University, USA; Thomas Schweder, Ernst-Moritz-Arndt-Universität Greifswald, Germany

                *Correspondence: Germán L. Rosano, Instituto de Biología Molecular y Celular de Rosario, Consejo Nacional de Investigaciones Científicas y Técnicas, Esmeralda y Ocampo, Rosario 2000, Argentina e-mail: rosano@ 123456ibr-conicet.gov.ar

                This article was submitted to Microbiotechnology, Ecotoxicology and Bioremediation, a section of the journal Frontiers in Microbiology.

                Article
                10.3389/fmicb.2014.00172
                4029002
                24478763
                68127ac6-3536-4a26-8ca2-be0fed11d4b1
                Copyright © 2014 Rosano and Ceccarelli.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 20 December 2013
                : 29 March 2014
                Page count
                Figures: 1, Tables: 2, Equations: 0, References: 217, Pages: 17, Words: 0
                Categories
                Microbiology
                Review Article

                Microbiology & Virology
                recombinant protein expression,escherichia coli,expression plasmid,inclusion bodies,affinity tags,e. coli expression strains

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