Antibody Laboratory, Protein Research Department, Genetic Engineering and Biotechnology Research Institute, City for Scientific Research and Technology Applications, Alexandria, Egypt.
Prep Biochem Biotechnol. 2012;42(5):426-47. doi: 10.1080/10826068.2011.637600.
Recombinant human consensus interferon-alpha (cIFN-α) was obtained by synthesizing a codon-optimized gene composed of the consensus nucleotides at each position in the human alpha interferon family and expressing it in Escherichia coli. The full cIFN-α gene was synthesized in two steps of assembly and amplification by polymerase chain reaction (PCR) using long (45-50 nucleotides) overlapped primers. The two-step PCR resulted in a DNA band of 504 base pairs (bp) corresponding to the calculated size of the cIFN-α gene. The synthetic gene was cloned into temperature-regulated Power3 expression vector. The ligated Power3-cIFN-α (Power3-cIFNα) plasmid carried the cIFN-α gene under transcriptional regulation of the heat-inducible λP(L) promoter. This expression system was optimized with respect to heat-shock temperature and time of induction in shake flask cultures. The produced cIFN-α protein was characterized by polyacrylamide gel electrophoresis and immunoassays. The majority of the expressed cIFN-α protein of about 19 kD in size accumulated in the form of inclusion bodies. After refolding and purification utilizing single-step ion-exchange chromatography on DEAE-Sepharose, the yield was 70 mg/L. cIFN-α anti-cancer activity was assayed and compared with the commercially available IFN-α 2a.
重组人广谱干扰素-α（cIFN-α）是通过合成一个由人类干扰素家族中每个位置的共有核苷酸组成的密码子优化基因，并在大肠杆菌中表达而获得的。全长 cIFN-α 基因通过聚合酶链反应（PCR）的两步组装和扩增合成，使用长（45-50 个核苷酸）重叠引物。两步 PCR 产生了一条 504 个碱基对（bp）的 DNA 带，与计算的 cIFN-α 基因大小相对应。合成基因被克隆到温度调节的 Power3 表达载体中。连接的 Power3-cIFN-α（Power3-cIFNα）质粒在热诱导的 λP（L）启动子的转录调控下携带 cIFN-α 基因。该表达系统在摇瓶培养中针对热休克温度和诱导时间进行了优化。所产生的 cIFN-α 蛋白通过聚丙烯酰胺凝胶电泳和免疫测定进行了表征。表达的 cIFN-α 蛋白约 19 kD 大小的大部分以包涵体的形式积累。经过一步离子交换层析在 DEAE-Sepharose 上复性和纯化后，产量为 70 mg/L。测定了 cIFN-α 的抗癌活性，并与市售的 IFN-α 2a 进行了比较。
