Production of biologically active light chain of tetanus toxin in Escherichia coli: Evidence for the importance of the C-terminal 16 amino acids for full biological activity

• Published in: FEBS letters 1993-06, Vol.323 (3), p.218-222
• Main Authors: Fairweather, Neil F., Sanders, Dagmar, Slater, Debbie, Hudel, Martina, Habermann, E., Weller, Ulrich
• Format: Article
• Language: English
• Subjects: Adrenal Medulla - drug effects; Adrenal Medulla - metabolism; Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Animals; Base Sequence; Biological and medical sciences; Calcium - metabolism; Cattle; Cells, Cultured; Cloning, Molecular; Clostridium tetani; DNA, Recombinant - metabolism; E. coli, Chromaffin cell; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Escherichia coli - genetics; Exocytosis; Fundamental and applied biological sciences. Psychology; Kinetics; Macromolecular Substances; Miscellaneous; Molecular Sequence Data; Mutagenesis, Site-Directed; Norepinephrine - metabolism; Oligodeoxyribonucleotides; Proteins; Recombinant protein; Recombinant Proteins - isolation & purification; Recombinant Proteins - toxicity; Restriction Mapping; Site directed mutagenesis; Tetanus toxin; Tetanus Toxin - genetics; Tetanus Toxin - isolation & purification; Tetanus Toxin - toxicity
• ISSN: 0014-5793; 1873-3468
• 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.