{Reference Type}: Journal Article
{Title}: Production of potent long-lasting consensus interferon using albumin fusion technology in Pichia pastoris expression system.
{Author}: Naseem MU;Ahmed N;Khan MA;Tahir S;Zafar AU;
{Journal}: Protein Expr Purif
{Volume}: 166
{Issue}: 0
{Year}: 02 2020
{Factor}: 2.025
{DOI}: 10.1016/j.pep.2019.105509
{Abstract}: Consensus interferon (cIFN) is a wholly synthetic therapeutic protein which is used to treat hepatitis C/B and certain types of malignancies. It has short serum half-life, therefore, to maintain its therapeutic level in the human body it requires thrice-weekly administration. Various strategies like PEGylation and micro-encapsulation have been developed during the last few years to enhance the pharmacokinetics of small therapeutic peptides. This study executed the human albumin-fusion technology, a simple and flexible approach to extend the serum circulating half-life of cIFN, because human serum albumin (HSA) has long circulating half-life (19 days) and very minute immunological activities. We integrated the codon-optimized HSA-cIFN fusion gene into Pichia pastoris genome by homologous recombination. The selection of hyper-resistant P. pastoris clone against Zeocin™ achieved a high-level secretory expression (250 mg/L) of fusion protein. HSA-cIFN fusion protein was purified using one-step purification by affinity chromatography with 34% recovery. The SDS-PAGE and SEC-HPLC analysis confirmed the final purified product has molecular weight of 87 kDa with 98% purity. Western blot analysis using anti-IFN antibodies further verified the purified HSA-cIFN fusion protein. The specific biological activity was 2.1 × 106 IU/mg as assessed by cytopathic inhibition assay, and half-life of fusion protein was estimated by in vitro thermal and proteolytic stability studies. This work concludes that by using albumin fusion technology, codon optimization and one-step purification a high yield of 86 mg/L of biologically active protein with improved serum half-life was obtained.