CORE--PEPTIDE SYNTHESIS AND ANTIBODY PRODUCTION

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The purpose of this Core is to provide synthetic peptides and polyclonal antisera required by the Projects of this Program to carry out structural, functional and mechanistic studies. The two PI's of this Core have collaborated for 25 years to design peptide immunogens and produce high affinity, high specificity antibodies towards peptides and proteins. Using the Merrifield automated solid phase approach to peptide synthesis, this Core will synthesize at the request of each PI of this Program Project a total of approximately 30 peptides (including phosphopeptides) per year. From among these 30 synthetic peptides or from expressed proteins, approximately seven will be selected every year as immunogens for the production of polyclonal antisera. Using the latest techniques for peptide/protein isolation and analysis, we would provide relatively large quantities (25 to 100 mg) of purified, fully characterized peptides. Preparative peptide purification will be achieved by reversed phase high performance liquid chromatography (RP- HPLC) in two solvent systems. Determination of purity will be accomplished using RP-HPLC, ion exchange chromatography on FPLC and capillary zone electrophoresis; characterization will use amino acid analysis, mass spectroscopy (MS) and Edman degradation when necessary. We will use conjugated synthetic peptides as immunogens to develop antibodies directed towards gene products predicted from the cDNA sequences or from amino acid sequencing of tryptic fragments of purified natural products. Peptides will be carefully designed in such a way that the same peptide can be coupled through a unique bond to a protein carrier or a chromatographic support to raise antibodies and facilitate their affinity purification. Alternatively, fusion proteins produced in bacteria or recombinant proteins using the baculovirus/insect cell system and provided by individual projects will be injected directly in rabbits for production of antisera. To reduce cross-reactivity with antigens other than the protein of interest, selected antisera will be immunoaffinity purified using the cognate peptide, recombinant protein, or fusion protein covalently bound to an agarose-based gel.