Novel Human Interferons Produced by Protein Engineering

IFN-亚型显示出与它们对IFN-α受体1（IFNAR 1）和2（IFNAR 2）的结合亲和力相关的不同生物活性。基于低能量模型，我们使用Rosetta程序设计了一种突变型IFN，其在位置163处包含从丝氨酸到谷氨酸的单个氨基酸改变。这种纯化的IFN突变蛋白显示出降低的抗病毒和抗增殖活性，表明163丝氨酸对IFN-α生物学功能是重要的。 我们将设计新的IFN-α构建体，同时考虑对IFNAR 2和IFNAR 1的结合亲和力。 我们还关注了14种天然干扰素α亚型（IFN-α 1a，2b，2c，4a，5，6，7a，8b，10a，14 c，16，17 b，21 a和21 b）。根据已发表的干扰素序列，将14种干扰素亚型的构建体克隆到酵母表达载体pKLAC 2中，然后转化到宿主菌株Yap 3中，其中编码酵母天冬氨酸蛋白酶3（YAP 3）的YAP 3基因被破坏。从培养物上清液收集分泌的IFN-α蛋白，并使用反相HPLC进一步纯化。初步数据显示，各种IFN-α具有广泛的生物活性，表明它们可能在生物体中发挥不同的生物学作用。纯化的干扰素亚型蛋白的进一步表征正在进行中，包括确定由不同病毒诱导的表达模式、由不同IFN调节因子调节的产生以及所使用的信号通路。这些信息将有助于合成其他新的基因工程干扰素。

IFN-α subtypes display different biological activities that associate with their binding affinities for the IFN-alpha receptor 1 (IFNAR1) and 2 (IFNAR2). Based on a low energy model, we used the Rosetta program to design a mutant IFN which contains a single amino acid change at position 163 from serine to glutamic acid. This purified IFN mutant protein showed decreased antiviral and antiproliferative activities, suggesting 163 serine is important for IFN-alpha biological functions. We are going to design new IFN-alpha constructs taking into consideration the binding affinity for both IFNAR2 and IFNAR1. We also focused on 14 natural interferon alpha subtypes (IFN-alpha 1a, 2b, 2c, 4a, 5, 6, 7a, 8b, 10a, 14c, 16, 17b, 21a, and 21b). Based on the interferon sequences published, 14 interferon subtype constructs were cloned into a yeast expression vector pKLAC2 and then transformed into a host strainYap3 in which the YAP3 gene encoding yeast aspartic protease 3 (YAP3) was disrupted. Secreted IFN-alpha proteins were collected from culture supernatants and further purified using reverse phase HPLC. Preliminary data showed that the various IFN-alphas exhibit a wide breadth and varied profile of biological activities, suggesting they may play distinct biological roles in living organisms. Further characterization of purified interferon subtype protein is ongoing, including determination of the expression patterns induced by different viruses, the production regulated by different IFN regulatory factor and the signal pathway used. This information will assist in the synthesis of other novel genetically engineered interferons.