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.

中文翻译：

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在巴斯德毕赤酵母表达系统中使用白蛋白融合技术生产强效持久的共有干扰素。

共识干扰素（cIFN）是一种完全合成的治疗性蛋白质，用于治疗丙型肝炎/乙型肝炎和某些类型的恶性肿瘤。它具有较短的血清半衰期，因此，要维持其在人体中的治疗水平，需要每周三次给药。在最近几年中，已开发出各种策略，例如PEG化和微囊化，以增强小型治疗性肽的药代动力学。这项研究执行了人类白蛋白融合技术，这是一种简单而灵活的方法，可以延长cIFN的血清循环半衰期，因为人类血清白蛋白（HSA）的循环半衰期很长（19天），并且免疫活性很小。我们通过同源重组将密码子优化的HSA-cIFN融合基因整合到巴斯德毕赤酵母基因组中。高抗性P的选择。针对Zeocin™的巴斯德毕赤酵母克隆实现了融合蛋白的高水平分泌表达（250 mg / L）。HSA-cIFN融合蛋白通过一步纯化通过亲和色谱法纯化，回收率为34％。SDS-PAGE和SEC-HPLC分析证实最终纯化的产物的分子量为87 kDa，纯度为98％。使用抗IFN抗体的蛋白质印迹分析进一步证实了纯化的HSA-cIFN融合蛋白。通过细胞病抑制试验评估其比生物学活性为2.1×106 IU / mg，并通过体外热和蛋白水解稳定性研究评估融合蛋白的半衰期。这项工作的结论是，通过使用白蛋白融合技术，密码子优化和一步纯化，可获得高产量的86 mg / L具有改善的血清半衰期的生物活性蛋白。