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 conjugated using the crosslinker, N-succinimidyl-3(2-pyridyldithio)-propionate. When purified, the conjugate was found to be heterogeneous as analyzed by SDS/PAGE, size-exclusion HPLC and reverse-phase HPLC. Therefore, we sought to synthesize a molecule that would be homogeneous and thus easier to characterize and evaluate its efficacy and toxicity for pharmaceutical drug development. A homogeneous chemical conjugate was successfully synthesized by using a mutant FGF-2 with Cys96 replaced by Ser ([S96]FGF-2) and a recombinant saporin mutant containing a single Cys at the -1 position (C-SAP). The latter was expressed in Escherichia coli and isolated to 99% purity by expanded-bed adsorption chromatography 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 homogeneous conjugate, designated as FGF2-C-SAP. The purified FGF2-C-SAP was more than 98% pure as judged by HPLC. In vitro biological assays indicated that FGF2-C-SAP was a potent inhibitor of protein synthesis in a cell-free system and was cytotoxic to FGF-receptor-bearing cells.