Production of Functional Recombinant Human Interferon-gamma by RTX CPD-fusion Technology

Abstract

Human interferon-gamma (hIFNγ) is an important pleiotropic cytokine with therapeutic application. Recombinant hIFNγ has been shown to be an effective pharmaceutical against a wide range of viral, immuno-suppressive diseases with promising prospects in cancer immunotherapy resulting in a strong increase in demand and price. The aim of this study was to develop an efficient method for production of soluble and active hIFNγ. To this end a fusion gene encoding hIFNγ and a C-terminally located His₁₀-tagged RTX CPD was constructed by two-step PCR. The fusion gene was cloned in an expression vector under inducible promoter and expressed in different E. coli strains such as Rosetta (appropriate for expression of eukaryotic genes containing rare codons), and two chaperone containing strains – BL21(DE3)/pG-KJE8 and BL21(DE3)/pG-Tf2. The transformed bacteria were cultivated at 24℃ to favour proper folding of the recombinant protein. Under these conditions the strain BL21(DE3)/pG-KJE8 was chosen as more suitable for production of soluble hIFNγ. The latter was purified by Ni²⁺- based affinity chromatography, the peak fractions were subjected to affinity chromatography combined with a direct on-column digestion to release hIFNγ, which was further purified by ion exchange chromatography. It is worth mentioning that this strategy, employing auto-cleaving and solubility-enhancing CPD tag, combined with on-column tag digestion was applied for the first time for production of hIFNγ.