We wish to establish a state-of-the-art Analytical Protein Microchemistry Facility. This will consist of a gas-phase sequenator and its attendant HPLC amino acid analyzer, an HPLC for protein and peptide purification, and peptide synthesis equipment. With the gas-phase sequenator, automated amino acid sequence analysis can be performed with very small amounts of material (less than 100 picomoles). For a typical protein this corresponds to 5-10 micrograms. These amounts are being produced by monoclonal antibody affinity chromatography or preparative two-dimensional gel electrophoresis in various laboratories of the user group. The proposed equipment will complement existing technical resources at the University including an Oligonucleotide Synthesizer, Cell Hybridoma Lab and Transmission Electron Microscope Facilities. It will serve active research programs in DNA and cDNA cloning, site directed mutagenesis, regulation of gene expression, and structural and functional characterization of proteins. In addition to the individual applications of each machine, a particular feature of these facilities is that they can be used effectively in conjunction. As examples, partial amino acid sequences will serve to define the possible sequences of an oligonucleotide hybridization probe, and these probes used in turn to isolate the genes encoding the initial protein. Conversely, from a DNA sequence one can unambiguously deduce the amino acid sequence. Peptides corresponding to parts of this sequence can be constructed, coupled to a carrier protein, and then used to generate antisera or monoclonal antibodies. These antibodies can be employed to characterize functionally the proteins by stimulating or blocking known functions. Finally, novel gene products may be identified using antibodies directed against peptides synthesized to correspond to open reading frames found by DNA sequence analysis. We envision these technologies complementing one another and the existing expertise in molecular biology, biochemistry, immunology, and tumor biology. They will allow a large number of investigators to address many fundamental questions of clinical and basic science.
