Escherichia coli is the organism 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 recombinant 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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Recombinant protein expression in Escherichia coli: advances and challenges.

http://wolfson.huji.ac.il/expression/local/rec-exp-99.pdf

Recombinant protein expression in Escherichia coli.

During the proteomics period, the growth in the use of recombinant proteins has increased greatly in the recent years. Bacterial systems remain most attractive due to low cost, high productivity, and rapid use. However, the rational choice of the adequate promoter system and host for a specific protein of interest remains difficult. This review gives an overview of the most commonly used systems: As hosts, Bacillus brevis, Bacillusmegaterium, Bacillussubtilis, Caulobacter crescentus, other strains, and, most importantly, Escherichia coli BL21 and E. coli K12 and their derivatives are presented. On the promoter side, the main features of the l-arabinose inducible araBAD promoter (PBAD), the lac promoter, the l-rhamnose inducible rhaPBAD promoter, the T7 RNA polymerase promoter, the trc and tac promoter, the lambda phage promoter pL, and the anhydrotetracycline-inducible tetA promoter/operator are summarized.

Overview of bacterial expression systems for heterologous protein production: from molecular and biochemical fundamentals to commercial systems.

Formation of the chromophore of green fluorescent protein (GFP) depends on the correct folding of the protein. We constructed a "folding reporter" vector, in which a test protein is expressed as an N-terminal fusion with GFP. Using a test panel of 20 proteins, we demonstrated that the fluorescence of Escherichia coli cells expressing such GFP fusions is related to the productive folding of the upstream protein domains expressed alone. We used this fluorescent indicator of protein folding to evolve proteins that are normally prone to aggregation during expression in E. coli into closely related proteins that fold robustly and are fully soluble and functional. This approach to improving protein folding does not require functional assays for the protein of interest and provides a simple route to improving protein folding and expression by directed evolution.

Rapid protein-folding assay using green fluorescent protein.

Pure, soluble and functional proteins are of high demand in modern biotechnology. Natural protein sources rarely meet the requirements for quantity, ease of isolation or price and hence recombinant technology is often the method of choice. Recombinant cell factories are constantly employed for the production of protein preparations bound for downstream purification and processing. Eschericia coli is a frequently used host, since it facilitates protein expression by its relative simplicity, its inexpensive and fast high density cultivation, the well known genetics and the large number of compatible molecular tools available. In spite of all these qualities, expression of recombinant proteins with E. coli as the host often results in insoluble and/or nonfunctional proteins. Here we review new approaches to overcome these obstacles by strategies that focus on either controlled expression of target protein in an unmodified form or by applying modifications using expressivity and solubility tags.

/pdf/soluble-expression-of-recombinant-proteins-in-the-cytoplasm-5c8yfblc2x.pdf

Soluble expression of recombinant proteins in the cytoplasm of Escherichia coli

http://compbio.korea.ac.kr/wiki/images/8/83/Mmbr.1996.expression.pdf

Savvas C. Makrides

Papers

Strategies for achieving high-level expression of genes in Escherichia coli.