A strategy for high-level expression of soluble and functional human interferon alpha as a GST-fusion protein in E. coli.
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.
Rabhi-Essafi
Imen
I
Molecular Biotechnology Group, Institute Pasteur, Tunis, Tunisia.
Sadok
Amine
A
Khalaf
Noureddine
N
Fathallah
Dahmani M
DM
D000595:Amino Acid Sequence; D001709:Biotechnology / Q000379:methods; D004926:Escherichia coli / Q000235:genetics*; D005982:Glutathione Transferase / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D007370:Interferon Type I / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D008969:Molecular Sequence Data; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D011994:Recombinant Proteins; D012995:Solubility; D013917:Thrombin / Q000737:chemistry
D007370:Interferon Type I; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D005982:Glutathione Transferase; D013917:Thrombin