INTRACELLULAR MODULATION OF SPECIFIC INTRACELLULAR PROTEINS

特定细胞内蛋白质的细胞内调节

• 批准号: 2464488
• 负责人: A RUSSO
• 金额: --
• 依托单位: DIVISION OF CLINICAL SCIENCES - NCI
• 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/2464488
• 关键词: antigen antibody reaction; binding proteins; chemical binding; chimeric proteins; enzyme activity; gene expression; genetic library; glucose 6 phosphate dehydrogenase; immunoglobulin genes; immunoglobulin structure; molecular site; mutant; nucleic acid sequence; posttranslational modifications; protein engineering; radiation protection; recombinant proteins; tissue /cell culture; transfection /expression vector; transposon /insertion element

Decreasing or eliminating the concentration and/or function of intracellular proteins post--translationally may obviate the need for or offer an alternative to the use of antisense constructs, as well as the use of 'knockout' technology. Moreover, if within an individual experiment one could eliminate an intracellular protein concentration and thereafter revert the cell to normalcy, a specific function of a given protein could be elegantly studied. The current project strives to accomplish just such an experimental system by altering the binding sites of heavy chain antibody antigen recognition sequences (HcFv) by random substitution within the nucleotide coding frames of the antigen determining regions (DR1/DR3). The recombinant constructs define novel binding proteins and also permit insertion of the recombinant(s) of interest into mammalian shuttle vectors having inducible promoters, such that a binding protein can be temporarily expressed intracellularly. A recombinant phage library expressing 10(12) different binding proteins on their surface as fusion proteins (fM 13p*-HcFv) have been constructed. A set of newly engineered recombinant proteins were isolated from the random library that specifically binds and eliminates the activity of glucose-6-phosphate dehydrogenase (G6PD). We chose one of the set of binding proteins and inserted its gene into a mammalian cell. Expression of the binding protein results in ablation of intracellular G6PD activity. Further, once induction has ceased, G6PD activity returns fully, i.e., a mutant and its internal revertant within the same cell has been constructed. The method is being adapted for use in binding oncogenic and viral proteins.

减少或消除的浓度和/或功能 细胞内蛋白质的表达可能会抑制细胞对蛋白质的需求， 提供了使用反义构建体的替代方案，以及 使用“淘汰”技术。此外，如果在单个实验中， 可以消除细胞内蛋白质浓度， 使细胞恢复正常，给定蛋白质的特定功能可以 优雅地学习。 目前的项目正努力实现这一目标 通过改变重链结合位点的实验系统 随机置换抗体抗原识别序列（HcFv） 在抗原决定区的核苷酸编码框内 (DR1/DR3）。 重组构建体定义了新的结合蛋白， 也允许将目的重组体插入哺乳动物细胞中 具有诱导型启动子的穿梭载体， 可以暂时在细胞内表达。 重组噬菌体文库 在其表面上表达10（12）种不同的结合蛋白作为融合蛋白 目的蛋白（fM 13p *-HcFv）的构建。 一套新设计的 从随机文库中分离重组蛋白， 特异性结合并消除葡萄糖-6-磷酸的活性 脱氢酶（G6PD）。 我们选择了一组结合蛋白， 将其基因插入哺乳动物细胞中。 结合的表达 蛋白导致细胞内G6PD活性的消除。 此外，一旦 诱导已经停止，G6PD活性完全恢复，即，一个突变体及其 同一细胞内的内部回复突变体已经构建。 述的方法 适用于结合致癌蛋白和病毒蛋白。