Bacterial expression of interferon alpha with new approaches to its PEGylation

The interferons are cytokines secreted from cells in response to different stimuli and have many biological effects including antiviral activity. For the past ten years PEGylated interferon alpha 2a and 2b (IFN), in combination with Ribavirin, have been the only therapies available for the patients suffering from chronic Hepatitis B and C viral infections. The challenges of producing IFN α2b for medical purposes are the high cost and time-consuming protein production in mammalian or plant cells. For the patients, frequent use of the drug increases the cost of the therapy. Human IFN is a glycosylated protein with a calculated molecular weight of about 19.4 kDa. It has been shown previously that the non-glycosylated form expressed in bacteria still retains its antiviral activity. However, overexpression of this eukaryotic protein in bacteria results in its cytoplasmic deposition as insoluble inclusion bodies that need to be solubilised and correctly folded. In this work, the gene encoding the full-length IFN was codon-optimized for bacterial expression and cloned in the expression vector pET30a under the control of the IPTG-inducible pT7 promoter. Protein expression was carried out at 37˚C in a 5 litre fermenter for 20hrs post-induction. The expressed protein was purified from the solubilised inclusion bodies and refolded by dialysis and its antiviral activity tested on human cell lines infected with HCMV. Addition of polyethylene glycol molecule (PEG) to IFN extends the half-life of the drug by maintaining a more effective concentration and therefore prolonged activity. Attempts were made in pegylating the purified IFN with branched as well as linear PEG and data will be presented on the results obtained.