title: 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.
authors.affiliation: PRIZM Pharmaceuticals, San Diego, CA 92121, USA.
authors.forename: Y J
authors.initials: YJ
authors.lastname: Buechler
authors.forename: B A
authors.initials: BA
authors.lastname: Sosnowski
authors.forename: K D
authors.initials: KD
authors.lastname: Victor
authors.forename: Z
authors.initials: Z
authors.lastname: Parandoosh
authors.forename: S J
authors.initials: SJ
authors.lastname: Bussell
authors.forename: C
authors.initials: C
authors.lastname: Shen
authors.forename: M
authors.initials: M
authors.lastname: Ryder
authors.forename: L L
authors.initials: LL
authors.lastname: Houston
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D006224:Cricetinae; D003432:Cross-Linking Reagents / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D017931:DNA Primers / Q000737:chemistry; D004228:Dithionitrobenzoic Acid / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D016222:Fibroblast Growth Factor 2 / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D006801:Humans; D000922:Immunotoxins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009699:N-Glycosyl Hydrolases; D010940:Plant Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D017354:Point Mutation / Q000235:genetics; D014176:Protein Biosynthesis; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D054789:Ribosome Inactivating Proteins, Type 1; D012270:Ribosomes / Q000378:metabolism; D000076985:Saporins; D013388:Succinimides / Q000737:chemistry
chemical_list: D003432:Cross-Linking Reagents; D017931:DNA Primers; D000922:Immunotoxins; D010940:Plant Proteins; D011994:Recombinant Proteins; D054789:Ribosome Inactivating Proteins, Type 1; D013388:Succinimides; D016222:Fibroblast Growth Factor 2; C018151:N-succinimidyl 3-(2-pyridyldithio)propionate; D004228:Dithionitrobenzoic Acid; D009699:N-Glycosyl Hydrolases; D000076985:Saporins; D003545:Cysteine