Expression and Purification of SAK-fused Human Interferon Alpha in Escherichia coli

Abstract

A method for improved refolding and purification of E. coli derived human Interferon -α (rhIFN α2b) from inclusion bodies as a Staphylokinase (SAK) fusion protein is described. Such a fusion protein did not require the supplementation of rare codons for expression and was found to be stable at 37 o C. The optimal conditions of refolding involved the use of a mild denaturating agent without the need for any other agents to prevent aggregation. The SAKrhIFN α2b fusion protein was successfully purified using two steps of purification and was cleaved using enterokinase into two fragments namely SAK and IFN. Both the proteins were found to be biologically active showing proper folding of both the fusion partners. The cleaved IFN showed similar retention time on RP-HPLC as the bacterial derived untagged purified IFN as well as similar molecular weight on Agilent 2100 Bioanalyzer indicating the right processing of the IFN after enterokinase cleavage. The expression levels of SAK-IFN were found to be two folds higher than that observed with untagged IFN under similar experimental conditions.

MOLECULAR CLONING AND HETEROLOGOUS EXPRESSION OF HUMAN INTERFERON ALPHA2b GENE

“…In addition, they may be of use for further molecular studies of hIFNα2b such as DNA-protein and protein-protein interactions to further elucidate their mechanism of action. When required, it is recommended to cleave the GST-tag from the fusion proteins by using a site-specific protease such as thrombin or factor Xa (Salunkhe et al, 2009).…”

“…The most challenging tasks in bacterial expression of foreign protein is the prevention of proteolytic degradation and accumulation of misfolded protein. Expression of proteolitically sensitive polypeptides by their fusion to other stable proteins has been shown to suppress degradation, although the mechanism responsible for the stabilization is not well understood (Salunkhe et al, 2009).…”

“…The hIFNα2b gene was isolated by using the standard PCR amplification technique (Salunkhe et al, 2009) using the isolated human genomic DNA as a template. A pair of specific primers was employed.…”

“…Interferon therapy is used (in combination with chemotherapy and radiation) as a treatment for many cancers, AIDS related Kaposi's sarcoma and chronic hepatitis B and C. More than half of hepatitis C patients treated with interferon respond with viral elimination (sustained virological response), better blood tests and better liver histology (Salunkhe et al, 2009).…”

Novel Techniques in the Production of Industrially Imperative Products

“…The expression of IFN-α cDNA was achieved directly in E.coli soon after it was first cloned. Since, E. coli could be grown to high cell densities, and strains used for recombinant protein production are generally regarded as safe, it is a preferred expression host for largescale fermentations [67].…”