Bis Regina L, Stauffer Tara M, Singh Surinder M, Lavoie Thomas B, Mallela Krishna M G
Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, 12850 E Montview Blvd, C238, Aurora, CO 80045, United States.
PBL Assay Science, 131 Ethel Road West, Suite 6, Piscataway, NJ 08854, United States.
Protein Expr Purif. 2014 Jul;99:138-46. doi: 10.1016/j.pep.2014.04.010. Epub 2014 Apr 30.
Interferon α-2a (IFNA2) is a member of the Type I interferon cytokine family, known for its antiviral and anti-proliferative functions. The role of this family in the innate immune response makes it an attractive candidate for the treatment of many viral and chronic immune-compromised diseases. Recombinant IFNA2 is clinically used to modulate hairy cell leukemia as well as hepatitis c. Historically, IFNA2 has been purified from human leukocytes as well as bacterial expression systems. In most cases, bacterial expression of IFNA2 resulted in inclusion body formation, or required numerous purification steps that decreased the protein yield. Here, we describe an expression and purification scheme for IFNA2 using a pET-SUMO bacterial expression system and a single purification step. Using the SUMO protein as the fusion tag achieved high soluble protein expression. The SUMO tag was cleaved with the Ulp1 protease leaving no additional amino acids on the fusion terminus following cleavage. Mass spectrometry, circular dichroism, 2D heteronuclear NMR, and analytical ultracentrifugation confirmed the amino acid sequence identity, secondary and tertiary protein structures, and the solution behavior of the purified IFNA2. The purified protein also had antiviral and anti-proliferative activities comparable to the WHO International Standard, NIBSC 95/650, and the IFNA2 standard available from PBL Assay Science. Combining the expression and purification protocols developed here to produce IFNA2 on a laboratory scale with the commercial fermenter technology commonly used in pharmaceutical industry may further enhance IFNA2 yields, which will promote the development of interferon-based protein drugs to treat various disorders.
干扰素α-2a（IFNA2）是I型干扰素细胞因子家族的成员，以其抗病毒和抗增殖功能而闻名。该家族在先天免疫反应中的作用使其成为治疗许多病毒和慢性免疫功能低下疾病的有吸引力的候选者。重组IFNA2在临床上用于调节毛细胞白血病以及丙型肝炎。历史上，IFNA2已从人白细胞以及细菌表达系统中纯化出来。在大多数情况下，IFNA2的细菌表达会导致包涵体形成，或者需要大量纯化步骤，从而降低了蛋白质产量。在这里，我们描述了一种使用pET-SUMO细菌表达系统和单一纯化步骤来表达和纯化IFNA2的方案。使用SUMO蛋白作为融合标签实现了高可溶性蛋白表达。SUMO标签用Ulp1蛋白酶切割，切割后在融合末端不留下额外的氨基酸。质谱、圆二色性、二维异核核磁共振和分析超速离心证实了纯化的IFNA2的氨基酸序列同一性、二级和三级蛋白质结构以及溶液行为。纯化的蛋白质还具有与世界卫生组织国际标准NIBSC 95/650以及PBL Assay Science提供的IFNA2标准相当的抗病毒和抗增殖活性。将此处开发的在实验室规模上生产IFNA2的表达和纯化方案与制药行业常用的商业发酵罐技术相结合，可能会进一步提高IFNA2的产量，这将促进基于干扰素的蛋白质药物治疗各种疾病的开发。
