INTRACELLULAR MODULATION OF SPECIFIC INTRACELLULAR PROTEINS

减少或消除对…的集中和/或作用 翻译后的细胞内蛋白可能不需要或 提供了一种使用反义构建物的替代方法，以及 “击倒”技术的使用。此外，如果在一个单独的实验中 可以消除细胞内的蛋白质浓度，然后 使细胞恢复正常，特定蛋白质的特定功能可以 被优雅地研究。目前的项目正努力实现这样的目标 一种改变重链结合位的实验系统 抗体抗原识别序列(HcFv)的随机替代 在抗原决定区域的核苷酸编码框内 (DR1/DR3)。这些重组构建体定义了新的结合蛋白和 还允许将感兴趣的重组(S)插入哺乳动物 具有可诱导启动子的穿梭载体，使得结合蛋白 可以暂时在细胞内表达。重组噬菌体文库的构建 融合表达10(12)种不同结合蛋白 构建了FM13P*-HcFv蛋白。一套新设计的 从随机文库中分离出重组蛋白 特异性地结合和消除葡萄糖-6-磷酸的活性 脱氢酶(G6PD)。我们从一组结合蛋白中选择了一种 将其基因植入哺乳动物细胞。绑定的表达 蛋白质导致细胞内G6PD活性的消融。更进一步，一次 诱导已经停止，G6PD活性完全恢复，即突变和其 已经在同一细胞内构建了内部回复突变体。方法 正在被改造用于结合致癌蛋白和病毒蛋白。

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.