Luo Sen, Li Tao, Wang Qin, Tian Renmao, Liu Hao, Fang Huali, Chen Fanghong, Wang Hui
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.
State Key Laboratory of Pathogens and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, PR China.
Anal Biochem. 2014 Oct 15;463:75-81. doi: 10.1016/j.ab.2013.06.019. Epub 2013 Jul 12.
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.
SNARE超家族有三个核心成员，即SNAP - 25、VAMP - 2和 syntaxin。SNAP - 25可被肉毒杆菌毒素（BoNTs）/A、/C和/E切割，而VAMP - 2是蛋白水解性BoNTs/B、/D、/F和/G的底物。在本研究中，我们构建了一个编码融合蛋白SNVP的杂交基因，该融合蛋白包含SNAP - 25的Met1至Gly206残基和VAMP - 2的Met1至Lys94残基。该杂交基因被克隆到携带N端pelB信号序列的原核载体中，并在大肠杆菌BL21(DE3) Rosetta中过表达。为了便于纯化该蛋白，设计了6×His双亲和标签作为融合蛋白的连接子和C端。通过在HisTrap FF柱上进行亲和层析将SNVP纯化至同质，经十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳测定其纯度超过97%。纯化蛋白的N端测序表明信号肽已成功去除。融合蛋白SNVP包含所有七种血清型BoNTs的蛋白酶切割位点。SNVP也被证明能被BoNTs的内肽酶（BoNT/A - LC、BoNT/B - LC、BoNT/E - LC和BoNT/G - LC）识别并切割。新型融合底物SNVP在最佳条件下表现出高生物活性，表明其有潜力用作BoNT检测试剂。
