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.
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.
Department of Cell Biology, Wellcome Foundation Ltd., Beckenham, Kent, UK.
N F
NF
Fairweather
D
D
Sanders
D
D
Slater
M
M
Hudel
E
E
Habermann
U
U
Weller
D000313:Adrenal Medulla / Q000187:drug effects / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004274:DNA, Recombinant / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009638:Norepinephrine / Q000378:metabolism; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000633:toxicity; D015183:Restriction Mapping; D013744:Tetanus Toxin / Q000235:genetics* / Q000302:isolation & purification / Q000633:toxicity*
D004274:DNA, Recombinant; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D013744:Tetanus Toxin; D002118:Calcium; D009638:Norepinephrine