Production of an expression system for a synaptobrevin fragment to monitor cleavage by botulinum neurotoxin B.
Recombinant DNA techniques were used to develop an expression system for a 51-amino acid peptide fragment that encompasses residues 44-94 of human synaptobrevin 2. This protein is associated with secretory vesicles of nerve terminals and is a substrate for four of the seven serotypes of botulinum neurotoxin (BoNT). The DNA for the recombinant peptide was amplified by the polymerase chain reaction and cloned into the pTrxFus vector. The resulting synaptobrevin peptide was expressed as a thioredoxin fusion protein in E. coli and released into the medium by osmotic lysis. The 18.7-kDa thioredoxin-synaptobrevin protein, designated as TSB-51, is intended for use in a cell-free assay to test potential inhibitors of BoNT/B-mediated proteolysis of synaptobrevin with the ultimate aim of developing clinically effective therapeutic agents to counteract botulism. Incubation of TSB-51 with the purified light chain of BoNT/B resulted in proteolysis which was evident within 30 min and increased with time until completion (approximately 4 hr). Cleavage of TSB-51 appeared to be at the appropriate BoNT/B cleavage site as indicated by a reduced intensity of the 18.7-kDa band and the appearance of a band at 16.4 kDa on Tris-tricene polyacrylamide gradient gels. The concentration of free Zn2+ had a significant effect on the cleavage rate; low Zn2+ concentrations stimulated substrate cleavage, whereas high concentrations were inhibitory. Cleavage was not significantly depressed by the naturally occurring metalloprotease inhibitor phosphoramidon when tested at concentrations up to 5 mM. TSB-51 appears to be a useful substrate for studying BoNT/B and is expected to aid in the discovery of effective BoNT inhibitors.
Nowakowski
J L
JL
Pharmacology Division, U. S. Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, Maryland 21010-5425, USA.
Courtney
B C
BC
Bing
Q A
QA
Adler
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D001905:Botulinum Toxins / Q000378:metabolism*; D019274:Botulinum Toxins, Type A; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D006868:Hydrolysis; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011480:Protease Inhibitors / Q000494:pharmacology; D050683:R-SNARE Proteins; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015032:Zinc / Q000378:metabolism
D004274:DNA, Recombinant; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D011480:Protease Inhibitors; D050683:R-SNARE Proteins; D011993:Recombinant Fusion Proteins; C096323:rimabotulinumtoxinB; D008666:Metalloendopeptidases; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A; D015032:Zinc