Camelid antibodies as sensitive proteomics tools for developmental studies

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DESCRIPTION (provided by applicant): Xenopus laevis is an important model organism for studies of vertebrate development. These studies are limited by lack of molecular tools that are necessary for following protein expression, localization and function during embryogenesis. This application will explore the utility of single domain antibodies (or nanobodies) from camelids for characterization and functional exploration of the Xenopus embryo proteome. In contrast to conventional monoclonal antibodies, which are expensive to generate and maintain, nanobodies are exceptionally stable and can be easily expressed in E. coli without loss of their binding activity. In our preliminary experiments, specific nanobodies against Xenopus antigens have been successfully isolated by sib-selection of a pilot cDNA library from llamas immunized with a complex gastrula embryo lysate. Based on these preliminary studies, we propose to develop a new class of antibody tools for the analysis of the Xenopus proteome. To achieve this goal, relevant nanobody cDNA libraries will be prepared and screened by expression cloning approaches to isolate nanobodies against specific Xenopus developmental antigens. These experiments should generate a number of useful molecular markers and will assess whether this approach can be extended to the Xenopus proteome and other vertebrate proteomes. These studies will also enable us to evaluate feasibility of nanobody expression cloning for functional embryological studies. By applying the nanobody technology to embryological studies, these experiments will have a significant impact on the future systems- level characterization and functional analysis of Xenopus embryonic development and will constitute a major help to the Xenopus community. The generated nanobodies will be used for the analysis of protein-protein interactions and gene regulatory networks in diverse developmental processes. The advanced knowledge of these processes is critical for the future understanding of mechanisms underlying human birth defects and the molecular causes of cancer.