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PEDS Advance Access originally published online on April 12, 2007
Protein Engineering Design and Selection 2007 20(5):201-209; doi:10.1093/protein/gzm012
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

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

Imen Rabhi-Essafi, Amine Sadok, Noureddine Khalaf and Dahmani M. Fathallah1

Molecular Biotechnology Group, Institute Pasteur, Tunis, Tunisia

1 To whom correspondence should be addressed. E-mail: dahmani.fathallah{at}pasteur.rns.tn; medfa{at}lycos.com

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} (rhIFN{alpha}) in E.coli, we have first constructed a recombinant expression plasmid (pGEX-hIFN{alpha}2b), in which we merged the hIFNFormula2b 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 rhIFN{alpha}2b 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}-hIFN{alpha}2b) and the modified E.coli trxB–/gor– (Origami) strain to overcome the problem of removing the GST moiety while expressing soluble rhIFN{alpha}2b. Our results show the production of soluble and functional rhIFN{alpha}2b at a yield of 100 mg/l, without optimization of any step of the process. The specific biological activity of the purified soluble rhIFN{alpha}2b was equal to 2.0 x 108 IU/mg when compared with the WHO IFN{alpha} standard. Our data are the first to show that high yield production of soluble and functional rhIFN{alpha}2b tagged with GST can be achieved in E.coli.

Keywords: E.coli/expression/interferon/recombinant/soluble

Received September 9, 2006; revised December 23, 2006; accepted January 3, 2007.


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