Fairweather N F, Sanders D, Slater D, Hudel M, Habermann E, Weller U
Department of Cell Biology, Wellcome Foundation Ltd., Beckenham, Kent, UK.
FEBS Lett. 1993 Jun 1;323(3):218-22. doi: 10.1016/0014-5793(93)81343-x.
The activity of the light (L) chain of tetanus toxin, and of mutants constructed by site-directed mutagenesis, was studied by expression and purification of the proteins from E. coli. Wild-type recombinant L chain (pTet87) was active in the inhibition of exocytosis from cultured bovine adrenal chromaffin cells, although at a level 5-15% of that of L chain purified from tetanus toxin. L chain mutants which terminated at Leu-438 (pTet89), or which contained a Cys-to-Ser mutation at residue 439 (pTet88) were equally as active as the full-length recombinant protein. The reduced activity of pTet87 L chain correlated with C-terminal proteolysis of the protein upon purification. A tryptic fragment derived from native light chain and which terminated at Leu-434 also showed reduced activity in the exocytosis assay, consistent with a requirement of the C-terminal region of the L chain for maximal activity. pTet87 L chain, but neither of the mutants, could be associated with purified H (heavy) chain to form a covalent dimer which induced the symptoms of tetanus in mice. The ability to form biologically active toxin using recombinant L chain will be of great value in structure-function studies of tetanus toxin.
通过从大肠杆菌中表达和纯化蛋白质，研究了破伤风毒素轻链（L链）及其通过定点诱变构建的突变体的活性。野生型重组L链（pTet87）在抑制培养的牛肾上腺嗜铬细胞的胞吐作用方面具有活性，尽管其活性水平仅为从破伤风毒素中纯化的L链的5%-15%。在Leu-438处终止的L链突变体（pTet89）或在残基439处含有半胱氨酸到丝氨酸突变的L链突变体（pTet88）与全长重组蛋白具有同等活性。pTet87 L链活性降低与纯化过程中该蛋白的C末端蛋白水解有关。源自天然轻链且在Leu-434处终止的胰蛋白酶片段在胞吐作用测定中也显示出活性降低，这与L链C末端区域对最大活性的需求一致。pTet87 L链，但不是这两个突变体中的任何一个，可与纯化的重链（H链）结合形成共价二聚体，该二聚体可在小鼠中诱发破伤风症状。使用重组L链形成生物活性毒素的能力在破伤风毒素的结构-功能研究中将具有重要价值。
