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Publication Details
AFRICAN RESEARCH NEXUS
SHINING A SPOTLIGHT ON AFRICAN RESEARCH
biochemistry, genetics and molecular biology
A strategy for high-level expression of soluble and functional human interferon α as a GST-fusion protein in E.coli
Protein Engineering, Design and Selection, Volume 20, No. 5, Year 2007
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Description
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 α (rhIFNα) in E.coli, we have first constructed a recombinant expression plasmid (pGEX-hIFNα2b), in which we merged the hIFNα2b 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 rhIFNα2b 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-Δ-hIFNα2b) and the modified E.coli trxB-/gor- (Origami) strain to overcome the problem of removing the GST moiety while expressing soluble rhIFNα2b. Our results show the production of soluble and functional rhIFNα2b at a yield of 100 mg/l, without optimization of any step of the process. The specific biological activity of the purified soluble rhIFNα2b was equal to 2.0 × 108 IU/mg when compared with the WHO IFNα standard. Our data are the first to show that high yield production of soluble and functional rhIFNα2b tagged with GST can be achieved in E.coli. © The Author 2007. Published by Oxford University Press. All rights reserved.
Authors & Co-Authors
Rabhi-Essafi, Imen
Tunisia, Tunis
Institut Pasteur de Tunis
Sadok, Amine
Tunisia, Tunis
Institut Pasteur de Tunis
Ben Khalaf, Noureddine
Tunisia, Tunis
Institut Pasteur de Tunis
Fathallah, Dahmani Mohamed
Tunisia, Tunis
Institut Pasteur de Tunis
Statistics
Citations: 91
Authors: 4
Affiliations: 1
Identifiers
Doi:
10.1093/protein/gzm012
ISSN:
17410126
e-ISSN:
17410134
Research Areas
Genetics And Genomics