Synthesis and Characterization of a Homogeneous Chemical Conjugate Between Basic Fibroblast Growth Factor and Saporin

Abstract

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. Copyright © 1995, Wiley Blackwell. All rights reserved