Novel Human Interferons Produced by Protein Engineering

蛋白质工程生产的新型人类干扰素

基本信息

批准号：
8336183
负责人：
Kathryn Zoon
金额：
$ 41.69万
依托单位：
NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
依托单位国家：
美国
项目类别：
财政年份：
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/8336183
关键词：
9p21 9p22 Adverse effects Affinity Amino Acid Sequence Amino Acids Antiviral Agents Aspartic Endopeptidases Binding Biological Biological Assay Biological Process Chimeric Proteins Chromosomes, Human, Pair 9 Data Engineering Exhibits Exons Gene Cluster Genes Glutamic Acid Goals High Pressure Liquid Chromatography Human Hybrids IFNAR1 gene Inflammatory Interferon Alfa-2a Interferon Type I Interferon-alpha Interferons Laboratories Lead Legal patent Leukocytes Life Modeling Molecular Models Nucleotides Open Reading Frames Organism Pattern Peptide Sequence Determination Phase Play Point Mutation Population Positioning Attribute Preparation Production Property Protein Engineering Proteins Publishing Role Sendai virus Serine Signal Pathway Variant Virus Yeasts base design expression vector molecular modeling mutant novel peripheral blood programs receptor

项目摘要

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 亚型表现出不同的生物活性，这与其与 IFN-α 受体 1 (IFNAR1) 和 2 (IFNAR2) 的结合亲和力相关。基于低能模型，我们使用Rosetta程序设计了一种突变型干扰素，其在163位上包含一个氨基酸从丝氨酸变为谷氨酸的突变体。这种纯化的 IFN 突变蛋白显示出抗病毒和抗增殖活性降低，表明 163 丝氨酸对于 IFN-α 的生物学功能很重要。我们将设计新的 IFN-α 构建体，考虑到 IFNAR2 和 IFNAR1 的结合亲和力。我们还关注 14 种天然干扰素 α 亚型（IFN-α 1a、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种干扰素亚型构建体克隆到酵母表达载体pKLAC2中，然后转化到宿主菌株Yap3中，其中编码酵母天冬氨酸蛋白酶3（YAP3）的YAP3基因被破坏。从培养物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-α 表现出广泛且多样的生物活性，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，包括确定不同病毒诱导的表达模式、不同干扰素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型基因工程干扰素。