Cloning, Expression, Purification, and Bio-functional Analysis of Human Interferon-γ
Immunoregulatory and anticancer functions are only a few examples of the function that are attributed to Human interferon-γ (hIFN-γ). In this study, we extracted the hIFN-γ gene from the peripheral T-lymphocytes of individuals. Next, the desired gene fragment was inserted into the inducible bacterial plasmid vector pET28a+ and the vector was eventually transformed into E. coli strain BL21. The recombinant hIFN-γ protein was expressed in different expression conditions using IPTG as expression inducer and the resultant inclusion bodies were solubilized using a previously described method based on pH fluctuation shock. The protein solution was refolded using a solution containing Tris–HCl, EDTA, urea, glycerol, sucrose, and PMSF and later on purified using Vivaflow 200, SP Sepharose Fast Flow. The functionality assay was performed on the recombinant hIFN-γ which eventually confirmed the success of the whole process. Our results finally demonstrated that the experimental procedures performed in this study are capable of large-scale production of the pharmaceutical valuable cytokine of interferon-γ.