title: Development of a fusion protein SNVP as substrate for assaying multi-serotype botulinum neurotoxins.
abstract: The SNARE super family has three core members, namely SNAP-25, VAMP-2, and syntaxin. SNAP-25 is cleaved by botulinum toxins (BoNTs)/A, /C, and /E, whereas VAMP-2 is the substrate for proteolytic BoNTs/B, /D, /F, and /G. In this study, we constructed a hybrid gene encoding the fusion protein SNVP that encompasses SNAP-25 residues Met1 to Gly206 and VAMP-2 residues Met1 to Lys94. The hybrid gene was cloned in a prokaryotic vector carrying an N-terminal pelB signal sequence and overexpressed in Escherichia coli BL21(DE3) Rosetta. To easily purify the protein, 6× His double-affinity tags were designed as the linker and C terminus of the fusion protein. SNVP was purified to homogeneity by affinity chromatography on a HisTrap FF column and determined to be more than 97% pure by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. N-terminal sequencing of the purified protein showed that signal peptide was successfully removed. The fusion protein SNVP contained the protease cleavage sites of all seven serotypes of BoNTs. SNVP was also proved to be recognized and cleaved by the endopeptidase of BoNTs (BoNT/A-LC, BoNT/B-LC, BoNT/E-LC, and BoNT/G-LC). The novel fusion substrate SNVP exhibited high biological activity under the optimal conditions, suggesting its potential use as a reagent for BoNT assay.
authors.affiliation: State Key Laboratory of Pathogens and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, PR China; Department of Microbiology, An Hui Medical University, An Hui 230032, PR China.
authors.forename: Sen
authors.initials: S
authors.lastname: Luo
authors.affiliation: State Key Laboratory of Pathogens and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, PR China.
authors.forename: Tao
authors.initials: T
authors.lastname: Li
authors.affiliation: State Key Laboratory of Pathogens and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, PR China.
authors.forename: Qin
authors.initials: Q
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Pathogens and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, PR China.
authors.forename: Renmao
authors.initials: R
authors.lastname: Tian
authors.affiliation: State Key Laboratory of Pathogens and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, PR China.
authors.forename: Hao
authors.initials: H
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Pathogens and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, PR China.
authors.forename: Huali
authors.initials: H
authors.lastname: Fang
authors.affiliation: State Key Laboratory of Pathogens and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, PR China.
authors.forename: Fanghong
authors.initials: F
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Pathogens and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, PR China. Electronic address: geno0109@vip.sina.com.
authors.forename: Hui
authors.initials: H
authors.lastname: Wang
mesh_terms: D001681:Biological Assay; D001905:Botulinum Toxins / Q000032:analysis* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D013379:Substrate Specificity; D050825:Synaptosomal-Associated Protein 25 / Q000235:genetics / Q000378:metabolism*; D050988:Vesicle-Associated Membrane Protein 2 / Q000235:genetics / Q000378:metabolism
chemical_list: D011993:Recombinant Fusion Proteins; D050825:Synaptosomal-Associated Protein 25; D050988:Vesicle-Associated Membrane Protein 2; D001905:Botulinum Toxins
keywords: Biological assay; Botulinum neurotoxin; SNAP-25; VAMP-2