PRODUCTION AND ANALYSIS OF A FIBROBLAST GROWTH FACTOR RECEPTOR UTILIZING THE BACULOVIRUS/INSECT CELL SYSTEM

Abstract

Fibroblast growth factors (FGFs) and their receptors (FGFRs) are known to play a role in Xenopus development. FGF-2 and FGFR-1 mRNA and protein are present in the oocyte and early embryo at the appropriate time and in sufficient concentration to be a mesodermal inducing factor in vivo. The purpose of this project was to produce and purify recombinant Xenopus FGFR- 1(rXFGFR-1) that was biologically and biochemically active. The Baculovirus/insect cell system was employed. Recombinant viral infection and protein production in infected insect cells were optimized. Antibodies raised against a peptide corresponding to XFGFR-1 were used to construct immunoaffinity columns for protein purification. Yield was 10Oug per 10⁸ infected insect cells. Functional rXFGFR-1 were found to be presented on the surface of infected insect cells and bound FGF-1 and FGF-2 specifically. Scatchard analysis of competitive binding studies revealed a dissociation constant of 1.5 nM with 3x10⁵ binding sites per cell. Kinase activity of the rXFGFR-1 was confirmed by immunoblotting cell extracts using an antiphosphotyrosine antibody, and autophosphorylation studies. Specificity of kinase activity was determined by phosphoamino acid analysis followed by thin layer chromatography. Post-translational modifications performed by the insect cells included signal peptide cleavage and glycosylation. The recombinant XFGFR-1 produced in the baculovirus/insect cell system has been shown to bind ligand, have functional and specific tyrosine kinase activity. The quantity of rXFGFR-1 produced and purified was adequate and the quality acceptable for further experimentation. Several lines of inquiry will be pursued involving library screening and other substrate searches, as well as ligand binding and receptor dimerization studies.