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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.

A modified pBAD24 vector (pBAD24M) was constructed with the araBAD promoter of the arabinose operon along with T7g10 sequence elements and a modified Shine-Dalgarno sequence. While both green fluorescent protein and granulocyte colony stimulating factor showed negligible expression under the original pBAD24 vector, they were expressed at >35% of total cellular protein with the modified vector. Similar results were obtained for staphylokinase wherein the pBAD24-SAK construct yielded 8 ng/10(6) c.f.u. of E. coli induced cells while the pBAD24M-SAK vector showed nearly 55 ng/10(6) c.f.u. induced bacterial cells as tested by ELISA. Interestingly, the expression levels using modified pBAD24 vector matched that achieved with T7 promoter based vector system. The modified pBAD24 vector therefore represents a simple and a useful prokaryotic expression system for efficient repression, modulation and elevated protein expression levels.

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