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

<p class="IJASEITAbtract"><span lang="EN-ID">Human i</span><span lang="EN-ID">nterferon alpha-2a (hIFNα-2a</span><span lang="EN-ID">) is a therapeutic protein that used in cancer and hepatitis B/C treatments.</span><span lang="EN-ID"> One main problem of using hIFNα-2a is the lack of good pharmacokinetic profile due to its low molecular weight. This research aims </span><span lang="EN-ID">to develop recombinant hIFNα</span>-<span lang="EN-ID">2a fusion protein by using human serum albumin</span> (HSA)<span lang="EN-ID"> to improve its molecular weight. </span>The<span lang="EN-ID"> codon optimized open reading frame (ORF) encoding fusion protein constructed synthetically and inserted </span><span lang="EN-ID">into pPICZαB expression vector and transformed into <em>Escherichia coli</em> XL1-blue. The characterized recombinant plasmid was linearized and transformed into methilotropic yeast <em>Pichia pastoris</em> 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 </span>the <span lang="EN-ID">recognition by anti HSA and anti hIFNα</span>-<span lang="EN-ID">2 antibodies. This result strongly indicated that the fusion protein was successfully produced in <em>Pichia pastoris. </em>Protein quantification informed that the protein yield was 14 mg/L (OD600=2). Stability expression analysis showed that protein production was stable until 60^th generations. </span>Preliminary a<span lang="EN-ID">ntiproliverative</span><span lang="EN-ID"> activity assay demonstrated that the fusion protein had 20% lower activity comparing to non fusion form. </span>