Cloning and expression of a region of vesicle associated membrane protein2 (VAMP2) gene and its use as a recombinant peptide substrate for assaying clostridial neurotoxins in contaminated biologicals.
An assay for the endopeptidase activities of clostridial neurotoxins in contaminated biotherapeutic products has been developed. Based on a synthetic peptide substrate representing amino acid residues 60-94 of the intracellular vesicle associated membrane protein2 (VAMP2), RT-PCR was used to amplify the VAMP2 sequence. The extended insert was digested with EcoRI and SalI and ligated into pGEX4T-1 vector for construction of the pGEX4T-1/VAMP plasmid for expressing in Escherichia coli a fusion protein linked to glutathione S-transferase (GST). The fusion protein was purified by affinity chromatography and used in an ELISA assay for comparison with the commercially available synthetic VAMP peptide and rabbit polyclonal antiserum. The identity of the immunoreactivity of recombinant VAMP2 protein with the chemically synthesized peptide was demonstrated by western blot. Our results indicated that recombinant VAMP2 peptide not only reacted with specific polyclonal antibody in a dose-dependent manner, without any remarkable difference observed between the reactivity of the fusion protein and commercial VAMP2 segment peptide, but also cleaved by botulinum neurotoxin type B (BONT/B) after endopeptidase assay. Thus, recombinant VAMP2 could serve as a replacement for VAMP2 synthetic peptide, potentially useful in endopeptidase assays for replacement of the currently used mouse bioassay for clostridial neurotoxins contaminating biotherapeutic products.
Moghaddam
Mehrdad Moosazadeh
MM
Applied Biotechnology & Environmental Research Center, Baqiyatallah Medical Science University, Tehran, Iran.
Mousavi
Latif
L
Shokrgozar
Mohammad Ali
MA
Amani
Jafar
J
Nazariyan
Shahram
S
Azari
Shahram
S
D000818:Animals; D001688:Biological Products / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism / Q000592:standards; D001905:Botulinum Toxins / Q000378:metabolism / Q000494:pharmacology; D019274:Botulinum Toxins, Type A; D003001:Cloning, Molecular; D004340:Drug Contamination; D010450:Endopeptidases / Q000378:metabolism; D005260:Female; D015870:Gene Expression; D005982:Glutathione Transferase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D051379:Mice; D008828:Microbiological Techniques; D009498:Neurotoxins; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D011914:Rats, Inbred BN; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013744:Tetanus Toxin / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism*; D050988:Vesicle-Associated Membrane Protein 2 / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
D001688:Biological Products; D009498:Neurotoxins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D013744:Tetanus Toxin; C494706:Vamp2 protein, rat; D050988:Vesicle-Associated Membrane Protein 2; C096323:rimabotulinumtoxinB; D005982:Glutathione Transferase; D010450:Endopeptidases; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A