Biochemical Studies on Recombinant Interferon Gamma Produced in E. coli

Abstract

Production of therapeutic proteins in prokaryotic system,Escherichia coli(E.coli);has beenrecognizedas an effective stagefor production of recombinant human interferon gamma (rhIFN-γ). Modificationof rhIFN-γ expression is a linethat can bepositively employed for increasing the yield of production through optimization of induction conditions in shaker flasks to be applied onto batch culture ofrecombinant E. coli. Hence, in this research induction conditions for the over-production of rhIFN-γ including type of media, pH, type and amount of inducer were optimized.The factors considered for optimized conditions of the recombinant E.coli were the use of growth medium LB, neutral pH7 and the inducer (lactose) at final concentration 2mM. These factors found to be useful in batch process development. The cell density was reached to 7gm/L wet cell weight after 12h of batch fermentation. Commonly, the recombinant proteins were produced in E. coli as insoluble aggregates called inclusion bodies (IBs).A method for purification and refolding of rhIFN-γ from IBshas beendesignated. It includessolubilization of IBs in 8M guanidinium hydrochloride; refolding of rhIFN-γ by rapid dilution method; and protein purificationbyHitrap Q XL strong anion chromatography. The rhIFN-γ obtained has been characterized by immunogenicity against the hIFN-γ antibodies.The specific activity of purified rhIFN-γ was 1.87 x 107 IU/mg compared to standard rhIFN-γ via new MxAreporter gene assay which depends on identifying MxA mRNA level using real time PCR. The rhIFN-γ increases expression of the MxA gene product in direct relation to the dose of rhIFN-γ in IU.