| Systematic identification and high-throughput generation of membrane protein targeting reagents such as antibodies have not been achieved even though the importance and diversity of plasma membrane proteins in cell targeting applications is known. The major goal of this thesis research was to develop an antibody-based approach to screen in vitro antibody libraries using the target cell proteome for simultaneous identification of antibodies and their cognate plasma membrane protein targets. To this end, we have developed two different modes of antibody library screening methods, both based on yeast display technology. In the first approach, a yeast display library of human single-chain antibody fragments (scFvs) was biopanned against a cultured layer of brain microvascular endothelial cell line, yielding 34 unique antibody sequences. The inherent low non-specific binding between the yeast cells and mammalian cells enabled rapid enrichment of the scFv clones. The target plasma membrane proteins recognized by the scFvs were characterized and identified using an immunoprecipitation method termed yeast display immunoprecipitation (YDIP) using the yeast cells expressing scFvs directly as an immunoaffinity reagent.;The second mode of antibody screening uses scFv-antigen interaction in detergent solutions to screen a yeast display human scFv library against a target cellular plasma membrane proteome directly using biotinylated detergent-solubilized lysates of the target cell by fluorescence-activated cell sorting. Using this approach, antibody clones that bind to target cell proteome were identified. This mode of antibody library screening is advantageous for the simultaneous identification of antibody-cognate target membrane protein pair, since the screening process is in principle identical to the YDIP procedure. In addition, cells obtained from tissue samples can be used for antibody identification in vivo. Moreover, the FACS-based screening process provides an opportunity to interrogate the target antigens, obtaining important biological information relevant to the cellular localization and function of target membrane proteins and potentially the antibody itself. Taken together, these methodologies have great potential for facile creation and characterization of targeting reagents against membrane proteins and the discovery of novel targets. |
