Novel Human Interferons Produced by Protein Engineering

干扰素亚型表现出不同的生物活性，这与它们与干扰素-α受体1(IFNAR1)和2(IFNAR2)的结合亲和力有关。基于低能模型，我们利用Rosetta程序设计了一个突变的干扰素，该干扰素的163位氨基酸由丝氨酸变为谷氨酸。纯化的干扰素突变体蛋白具有较低的抗病毒和抗增殖活性，提示163丝氨酸对干扰素-α的生物学功能具有重要作用。考虑到IFNAR2和IFNAR1的结合亲和力，我们将设计新的干扰素-α构建体。 我们还重点研究了14种天然干扰素α亚型(干扰素-α1a、2b、2c、4a、5、6、7a、8b、10a、14c、16、17b、21a和21b)。根据已发表的干扰素序列，将14个干扰素亚型结构克隆到酵母表达载体pKLAC2中，转化酵母天冬氨酸蛋白酶3(YAP3)基因缺失的宿主菌株YAP3。从培养上清液中收集分泌的干扰素-α蛋白，用反相高效液相色谱法进一步纯化。初步数据显示，各种干扰素-α具有广泛的生物学活性，表明它们可能在生物体内发挥着不同的生物学作用。对纯化的干扰素亚型蛋白的进一步鉴定正在进行中，包括确定不同病毒诱导的表达模式、不同干扰素调节因子调节的产物以及所使用的信号通路。这些信息将有助于合成其他新型的基因工程干扰素。

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.