CATALYTIC ANTIBODIES FOF GAR TRANSFORMYLATION

这项提案概述了开发催化抗体的战略。 GAR转化。营养缺乏性互补将被用于 筛查催化抗体，因为筛查是基于催化 活跃性和非约束性。由于GARTfase的底物位于 细胞质，将有必要表达组合文库 (来自用多底物抑制剂免疫的小鼠 β-TGDF)。日本血吸虫功能性抗体的产生 利用硫氧还蛋白还原酶和硫氧还蛋白还原酶的突变体将促进细胞质的形成 利用在细胞质中表达的抗体优化orotate 本科维奇博士的实验室以前描述的是脱羧基。这个 最近体内的紧急情况(功能性抗体表达于 真核细胞质)作为治疗分子展示了 探索功能性抗体生产的重要性 细胞质。GARTfase催化抗体将有益于 表征了GAR转化的酶学性质。这 这些知识将有助于抗肿瘤药物的设计 抑制GARTfase。除了这一重要贡献之外， 该项目将产生催化抗体，酶的性质是琼脂 转化和细胞质抗体的产生，这个项目 会让申请者在组合图书馆有宝贵的经验 构建，基因组操作，催化抗体策略， 酵母菌的酶特性、抗体表达及优化 大肠埃希菌。

This proposal outlines a strategy for developing catalytic antibodies to GAR transformylation. Auxotrophic complementation will be used to screen for catalytic antibodies since the screening is based on catalytic activity and not binding. Since the substrates for GARTfase reside in the cytoplasm, it will be necessary to express the combinatorial library (derived from a mouse immunized with the multisubstrate inhibitor beta-TGDF) in the cytoplasm. Functional antibody production in the cytoplasm will be facilitated using mutants in thioredoxin reductase and optimized using an antibody expressed in the cytoplasm for orotate decarboxylation previously characterized in Dr. Benkovic's lab. The recent emergency of intrabodies (functional antibodies expressed in the eukaryotic cytoplasm) as therapeutic molecules demonstrates the importance of exploring functional antibody production in the cytoplasm. GARTfase catalytic antibodies will be beneficial for characterizing the enzymatic nature of GAR transformylation. This knowledge will be useful in the design of anti-neoplastic agents to inhibit GARTfase. In addition to the important contributions this project wold make to catalytic antibodies, the enzymatic nature f GAR transformylation, and cytoplasmic antibody production, this project would allow the applicant valuable experience in combinatorial library construction, genomic manipulations, catalytic antibody strategies, enzyme characterization, and antibody expression and optimization in E. coli.