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.
| Ostermeier, M; Benkovic, S J (2000) A two-phagemid system for the creation of non-phage displayed antibody libraries approaching one trillion members. J Immunol Methods 237:175-86 |
