Production of potent long-lasting consensus interferon using albumin fusion technology in Pichia pastoris expression system

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.
Source: Protein Expression and Purification - Category: Biochemistry Source Type: research