Tonello F, Pellizzari R, Pasqualato S, Grandi G, Peggion E, Montecucco C
Dipartimento di Scienze Biomediche, Università di Padova, Padova, I-35121, Italy.
Protein Expr Purif. 1999 Mar;15(2):221-7. doi: 10.1006/prep.1998.1007.
Tetanus neurotoxin (TeNT) consists of two disulfide-linked polypeptide chains, heavy (H) and light (L). The L chain is a zinc endopeptidase protein highly specific for vesicle-associated membrane protein (VAMP), which is an essential component of the exocytosis apparatus. Here we describe the cloning of the L chain of TeNT from Clostridium tetani strain Y-IV-3 (WS 15) and its expression in Escherichia coli as a glutathione S-transferase fusion protein. The full-length recombinant L chain, corresponding to residues 1-457, was obtained as a mixture of proteins of slightly different mass with identical N-terminal ends. To obtain a product useful for structural analysis and crystallization, a COOH-terminally truncated L chain (residues 1-427) was cloned, expressed, and purified with high yield. This truncated L chain is more active than the full-length and wild-type proteins in the hydrolysis of VAMP. Preliminary experiments of crystallization of the truncated recombinant L chain gave encouraging results.
破伤风神经毒素（TeNT）由两条通过二硫键连接的多肽链组成，即重链（H）和轻链（L）。轻链是一种锌内肽酶蛋白，对囊泡相关膜蛋白（VAMP）具有高度特异性，VAMP是胞吐装置的重要组成部分。在此，我们描述了从破伤风梭菌Y-IV-3菌株（WS 15）中克隆TeNT轻链，并将其作为谷胱甘肽S-转移酶融合蛋白在大肠杆菌中表达。对应于1至457位残基的全长重组轻链以具有相同N末端的质量略有不同的蛋白质混合物形式获得。为了获得可用于结构分析和结晶的产物，克隆、表达并高产纯化了一个羧基末端截短的轻链（1至427位残基）。该截短的轻链在VAMP水解中比全长和野生型蛋白更具活性。截短的重组轻链结晶的初步实验取得了令人鼓舞的结果。
