PRODUCTION AND CHARACTERIZATION OF AN ANALOG OF ACIDIC FIBROBLAST GROWTH-FACTOR WITH ENHANCED STABILITY AND BIOLOGICAL-ACTIVITY

We have used recombinant DNA methods to produce two forms of bovine ac idic fibroblast growth factor (aFGF), one with alanine substituted for the cysteine at position 47 and the other with the Ala47 change plus the substitution of glycine for the naturally occurring histidine at p osition 93. Both forms were expressed at high levels in Escherichia co li and purified to near homogeneity by solubilization of the inclusion bodies containing the aFGF, ion exchange chromatography, refolding of the protein and hydrophobic interaction chromatography. Circular dich roic and infrared spectra suggested that the proteins are similar in s econdary and tertiary structures and contain little or no alpha-helica l conformations. Hydrophobic interaction chromatography showed that aF GF C47A/H93G is slightly more hydrophobic than the aFGF C47A form, sug gesting that residue 93 is exposed to the solvent. Half-maximal activi ty in an in vitro bioassay system was reached at a 10- to 20-fold lowe r dose for the aFGF C47A/H93G form than for the aFGF C47A form, sugges ting that alteration of this residue has an effect on the region respo nsible for receptor binding. Addition of 50 mug/ml heparin enhanced th e in vitro activity of the aFGFs, reducing the half-maximal dose to ap proximately 100 pg/ml for both forms, comparable to that observed prev iously for basic FGF with or without heparin in this assay system.