Artificial hybrid protein containing a toxic protein fragment and a cell membrane receptor-binding moiety in a disulfide conjugate. I. Synthesis of diphtheria toxin fragment A-S-S-human placental lactogen with methyl-5-bromovalerimidate.
In order to study the mechanism of entry of plant seed and bacterial toxins into mammalian cells, methods have been developed to synthesize artificial protein hybrid conjugates containing a moiety which binds to a cell membrane receptor and an active fragment of a toxin protein. Utilizing methyl-5-bromovalerimidate, a disulfide cross-linked conjugate of human placental lactogen (hPL) and diphtheria toxin fragment A (toxin A) was synthesized. The reagent was prepared from 5-bromovaleryl nitrile by Pinner synthesis and then used to amidinate hPL. The bromo group thus introduced was converted to S-sulfonate by nucleophilic displacement with 1 M aqueous sodium thiosulfate at room temperature overnight. The S-sulfonated hPL reacted readily with the-SH gorup of reduced toxin A to form a 1 mol/mol of disulfide conjugate in high yield. Thus when reduced toxin A was incubated with a 4-fold excess of the hPL S-sulfonate at 4 degrees and pH 6.5 for 120 h, a conjugate yield of 50% relative to the toxin A input was obtained. Homopolymer formation was negligible and the product was purified by gel filtration on Sephadex G-150. Purity of the conjugate estimated by quantitative analysis of sodium dodecyl sulfate gels was 90%. The toxin A-hPL conjugate retained the activities of both toxin A and hPL, as reported in the accompanying paper. This method of preparing protein hybrid conjugates appeared to have advantages over previous methods utilizing bifunctional reagents with respect to both yield and freedom from homopolymer formation.
D000818:Animals; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D004167:Diphtheria Toxin; D004591:Electrophoresis, Polyacrylamide Gel; D007094:Imides; D007096:Imidoesters; D046911:Macromolecular Substances; D008970:Molecular Weight; D010446:Peptide Fragments; D010928:Placental Lactogen; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000378:metabolism; D011955:Receptors, Drug
D004167:Diphtheria Toxin; D007094:Imides; D007096:Imidoesters; D046911:Macromolecular Substances; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D011955:Receptors, Drug; C014091:methyl-5-bromovalerimidate; D010928:Placental Lactogen