Soluble expression of recombinant proteins in the cytoplasm of Escherichia coli

Abstract

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.

Effects of Environmental Factors on Soluble Expression of a Humanized Anti-TNF-α scFv Antibody in Escherichia coli

“…17,21,24 Inclusion bodies are misfolded proteins due to inefficient interaction between nascent protein and chaperone molecules which causes unsuitable folding of protein. 30 Some protocols have been developed based on environmental modifications to produce the soluble form of the expressed proteins in E. coli.…”

“…11,21 As mentioned above, the expression of scFv antibodies in E. coli cytoplasm generally leads to the production of inclusion bodies, 17,[22][23][24] so optimized growth conditions are required to achieve soluble expression of the target scFv. Therefore, in this study, we have examined the effects of temperature, shaking condition, concentration of inducers (lactose or IPTG), incubation time, pH, sucrose and Mg 2+ concentrations, and osmotic or heat shocks on the soluble expression of a 52 kD GST-fusion recombinant protein called GST-hD2 scFv in Escherichia coli.…”

Exploring three different expression systems for recombinant expression of globins: Escherichia coli, Pichia pastoris and Spodoptera frugiperda

“…Aggregation prone proteins require the existence of a number of molecular chaperones that interact reversibly with nascent polypeptide chains to prevent aggregation during the folding process. The formation of inclusion bodies could therefore result either from accumulation of high concentrations of folding intermediates or from inefficient processing by molecular chaperones [23]. Refolding from inclusion bodies is in many cases considered undesirable as it results most of the time in a poor recovery of bio-active protein [24].…”

“…Most used strategies for shifting the recombinant expression from inclusion bodies to soluble protein are: changing the expression temperature, media and transcription rates, trying various E.coli strains, including molecular chaperones, including tRNA complementation plasmids, expressing fragments, using fusion technology, stabilizing mRNA or screening for soluble variants. For further reading on these strategies we refer to the review paper of Sorensen et al [23].…”

Novel GFP Expression Using a Short N-Terminal Polypeptide through the Defined Twin-Arginine Translocation (Tat) Pathway

“…coli is frequently used as a host for recombinant protein expression because of its relatively simple, quick, and inexpensive cultivation, its well-characterized genetics, and the availability of a large number of useful molecular tools (Sørensen and Mortensen, 2005). Despite these merits, eukaryotic cell systems are gaining favor for the production of more complex recombinant proteins, such as glycosylated proteins or those with complex folding characteristics.…”