Each laboratory will continue purification of their respective growth factors using HPLC methodologies to obtain enough homogeneous material suitable for gas phase microsequencing of N-termini. Production of adequate amounts of starting material from conditioned medium will be done by mass cell culture techniques and selection of high growth factor producers. For the next year, as each of the laboratories make progress in isolation of factors, they will intensify their interactions with the protein core laboratory and the recombinant DNA laboratory. Each laboratory will attempt to obtain partial N-terminal amino acid sequences for their factors and prepare oligonucleotide cDNA probes based on the peptide sequence information by automated DNA synthesis. The probes will be used to isolate recombinant bacterial clones that contain DNA sequences coding for the factor. The complete amino acid sequence of the growth factors will be determined by deduction after sequencing the DNA coding for it. Knowledge of amino acid sequences will be used to prepare synthetic peptides suitable for production of high-affinity polyclonal or monoclonal antibodies for development of radioimmunoassays. Peptide synthesis will be carried out by the Merrifield method using automated synthesizers. (V)
Showing the most recent 10 out of 109 publications
