RELATIONSHIP OF STRUCTURE & FUNCTION OF HU IFN-GAMMA BY SITE-DIRECTED MUTAGENESIS

结构关系

• 批准号: 3811195
• 负责人: R Q HU
• 金额: --
• 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/3811195
• 关键词: Escherichia coli; gel electrophoresis; immunoprecipitation; interferon inducers; interferons; molecular cloning; mutant; polymerase chain reaction; protein engineering; protein structure function; site directed mutagenesis; transposon /insertion element

It has been shown that deletion of residue -1 to 7 or 9 residues of rhu IFN -gamma results in the loss of biological activity (H. Hogrefe et al ref. J. Biol. Chem. 21, 12179-86, 1989). Due to the limited amount of material that can be isolated by enzymatic modification, we are using a protein engineering approach to study the relationship between the structure and function of human IFN-gamma. The human IFN-gamma gene has been cloned into PGEM 2 vector and PKK223-3 IFN-gamma gene expressing vector has been constructed. Immunoprecipitation assay and SDS PAGE revealed that cells induced by IPTG produced human IFN-gamma in E. coli JM105. Using PCR and oligonucleotide directed mutagenesis, several mutant genes which contain truncate or replacement at N-terminal will be obtained. Some mutant genes will be cloned into the expression vector, and characterized.

已经表明，缺失rhu IFN的-1至7或9个残基， - γ导致生物活性的丧失（H. Hogrefe等人参考文献J. Biol. 21，12179-86，1989）。由于材料有限， 可以通过酶促修饰分离，我们使用的是一种蛋白质， 工程方法来研究结构和 人IFN-γ的功能。人IFN-γ基因已被克隆到 PGEM 2载体和PKK 223 -3 IFN-γ基因表达载体已被成功地克隆。 构建了免疫沉淀法和SDS PAGE显示， 在E. coli JM 105。 利用PCR和寡核苷酸定向突变技术， 将获得在N-末端含有截短或替换的蛋白。一些 将突变基因克隆到表达载体中并进行表征。