Development of Higher Molecular Weight of Recombinant Human Interferon Alpha-2a by Albumin Fusion Technology in Methilotropic Yeast Pichia Pastoris

Human interferon alpha-2a (hIFNα-2a) is a therapeutic protein that used in cancer and hepatitis B/C treatments. One main problem of using hIFNα-2a is the lack of good pharmacokinetic profile due to its low molecular weight. This research aims to develop recombinant hIFNα-2a fusion protein by using human serum albumin (HSA) to improve its molecular weight. The codon optimized open reading frame (ORF) encoding fusion protein constructed synthetically and inserted into pPICZαB expression vector and transformed into Escherichia coli XL1-blue. The characterized recombinant plasmid was linearized and transformed into methilotropic yeast Pichia pastoris GS115 and SMD 1168. The expression analysis showed that the best expression was achieved by protease deficient strain SMD1168. Molecular weight characterization informed that the fusion protein was 85 kDa in size as its theoritical size. Western blotting methods confirmed the identity of fusion protein based on the recognition by anti HSA and anti hIFNα-2 antibodies. This result strongly indicated that the fusion protein was successfully produced in Pichia pastoris. Protein quantification informed that the protein yield was 14 mg/L (OD600=2). Stability expression analysis showed that protein production was stable until 60th generations. Preliminary antiproliverative activity assay demonstrated that the fusion protein had 20% lower activity comparing to non fusion form.