Novel Human Interferons Produced by Protein Engineering

蛋白质工程生产的新型人类干扰素

• 批准号: 8156962
• 负责人: Kathryn Zoon
• 金额: $ 40.58万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8156962
• 关键词: Human; Interferons; Protein Engineering; novel

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.

IFN-α亚型显示出不同的生物学活性，这些活性与IFN-Alpha受体1（IFNAR1）和2（IFNAR2）的结合亲和力相关联。基于低能模型，我们使用Rosetta程序设计了一个突变体IFN，其中包含从丝氨酸到谷氨酸的位置163处的单个氨基酸变化。这种纯化的IFN突变蛋白显示出抗病毒和抗增生活性的降低，这表明163丝氨酸对于IFN-Alpha生物学功能很重要。 我们将设计新的IFN-Alpha构建体，考虑到IFNAR2和IFNAR1的结合亲和力。 我们还专注于14个天然干扰素α亚型（IFN-Alpha 1a，2b，2c，4a，5，6，6，7a，8b，8b，10a，14c，14c，16，16，17b，21a和21a和21a）。基于发表的干扰素序列，将14个干扰素亚型构建体克隆到酵母表达载体pKLAC2中，然后转化为宿主的过氧基3，其中YAP3基因编码酵母菌天然病蛋白酶3（YAP3）被干扰。从培养上清液中收集分泌的IFN-α蛋白，并使用反相HPLC进一步纯化。初步数据表明，各种IFN-ALLASE都表现出广泛的广度和不同的生物学活动特征，这表明它们可能在生物体中起着不同的生物学作用。纯化的干扰素亚型蛋白的进一步表征正在进行中，包括确定不同病毒引起的表达模式，由不同的IFN调节因子调节的产生和所使用的信号途径。该信息将有助于综合其他新型基因工程干扰素。