A strategy for high-level expression of soluble and functional human interferon α as a GST-fusion protein in E.coli

Abstract

Escherichia coli is the most extensively used host for the production of recombinant proteins. However, most of the eukaryotic proteins are typically obtained as insoluble, misfolded inclusion bodies that need solubilization and refolding. To achieve high-level expression of soluble recombinant human interferon alpha (rhIFNalpha) in E. coli, we have first constructed a recombinant expression plasmid (pGEX-hIFNalpha2b), in which we merged the hIFNalpha2b cDNA with the glutathione S-transferase (GST) coding sequence downstream of the tac-inducible promoter. Using this plasmid, we have achieved 70% expression of soluble rhIFNalpha2b as a GST fusion protein using E. coli BL21 strain, under optimized environmental factors such as culture growth temperature and inducer (IPTG) concentration. However, release of the IFN moiety from the fusion protein by thrombin digestion was not optimal. Therefore, we have engineered the expression cassette to optimize the amino acid sequence at the GST-IFN junction and to introduce E. coli preferred codon within the thrombin cleavage site. We have used the engineered plasmid (pGEX-Delta-hIFNalpha2b) and the modified E. coli trxB(-)/gor(-) (Origami) strain to overcome the problem of removing the GST moiety while expressing soluble rhIFNalpha2b. Our results show the production of soluble and functional rhIFNalpha2b at a yield of 100 mg/l, without optimization of any step of the process. The specific biological activity of the purified soluble rhIFNalpha2b was equal to 2.0 x 10(8) IU/mg when compared with the WHO IFNalpha standard. Our data are the first to show that high yield production of soluble and functional rhIFNalpha2b tagged with GST can be achieved in E. coli.

Strategies for production of active eukaryotic proteins in bacterial expression system

“…The most popular affinity tags are poly-histidine (His6) tags, which are compatible with immobilized metal affinity chromatography (IMAC) and the glutathione S-transferase (GST) tag for purification through glutathione based resins. GST, 27 kDa, can be prohibitive due to the slow binding kinetics of GST to glutathione-sepharose resin and lead to loading of cell extracts extremely time consuming, especially when large cell culture volumes are being processed. Poly-histidine tags, on the other hand, are small and do not, in most cases, affect the folding of the attached protein.…”

“…It also has very strong reversible binding attributes allowing for rapid single-step purification. Polyhistidine tags can be attached on either the N-or C-termini of recombinant proteins, but the optimal location depends on the folding and biochemical characteristics of the adjacent recombinant protein [14,20,26,27] .…”

Soluble Prokaryotic Expression and Purification of Human Interferon Alpha-2b Using a Maltose-Binding Protein Tag

“…Several factors have made the E. coli recombinant protein expression system favorable for the production and purification of IFNα. IFNα genes do not have introns and many of the protein products are active without O -glycosylation [Asmana Ningrum, 2014;Li et al, 2013a;Rabhi-Essafi et al, 2007]. In addition, E. coli can grow rapidly and to high cell densities, enabling efficient large-scale protein production.…”

Expression and purification of GST-FHL2 fusion protein

“…The high speed of protein synthesis at these temperatures makes fusion body formation likely to occur. We reasoned by lowering the temperature (Rabhi-Essafi et al, 2007) it would slow the rate of protein synthesis and promote correct protein folding over the formation of inclusion bodies, thereby increasing soluble protein expression. Therefore, we decreased the induction temperature to 23°C, and extended the induction time, so that the foreign protein could express sufficiently.…”