SYNTHESIS AND CHARACTERIZATION OF A HOMOGENEOUS CHEMICAL CONJUGATE BETWEEN BASIC FIBROBLAST GROWTH-FACTOR AND SAPORIN

Basic fibroblast growth factor (FGF-2) and saporin were chemically con jugated using the crosslinker, N-succinimidyl-3(2-pyridyldithio)-propi onate. When purified, the conjugate was found to be heterogeneous as a nalyzed by SDS/PAGE, size-exclusion HPLC and reverse-phase HPLC. There fore, we sought to synthesize a molecule that would be homogeneous and thus easier to characterize and evaluate its efficacy and toxicity fo r pharmaceutical drug development. A homogeneous chemical conjugate wa s successfully synthesized by using a mutant FGF-2 with Cys96 replaced by Ser ([S96]FGF-2) and a recombinant saporin mutant containing a sin gle Cys at the -1 position (C-SAP). The latter was expressed in Escher ichia coli and isolated to 99% purity by expanded-bed adsorption chrom atography followed by cation-exchange chromatography. The cysteine in C-SAP was activated by Ellman's reagent and then reacted with the only available cysteine (position 78) in [S96]FGF-2 to produce the homogen eous conjugate, designated as FGF2-C-SAP. The purified FGF2-C-SAP was mon than 98% pure as judged by HPLC. In vitro biological assays indica ted that FGF2-C-SAP was a potent inhibitor of protein synthesis in a c ell-free system and was cytotoxic to FGF-rrceptor-bearing cells.