EXPRESSION AND MUTAGENESIS OF RECOMBINANT CHOLERA-TOXIN-A SUBUNIT

ADP-ribosylating protein exotoxins from Vibrio cholerae (CT) and Esche richia coil (LT-I) share two short regions of sequence similarity with Bordetella pertussis toxin (PT). Previous studies have indicated that substitution of arginine for lysine 7 within the first region of CT d rastically decreases ADP ribosyltransferase activity. We have more clo sely defined the role of other amino acids in this region by generatin g modified proteins in which arginine 7 was replaced with lysine (R7K) , aspartate 9 was replaced with arginine (D9R), glycine was substitute d for proline 12 (P12G), amino acids 6 to 13 were deleted (Delta 613) or the C-terminal KDEL sequence was changed to NEDL. The modified prot eins R7K, D9R and Delta 613 exhibited undetectable ADP ribosyltransfer ase activity. Comparison of the tryptic digest of R7K with native CT s uggested that changes in protein conformation may be responsible for t he loss of ADP-ribosylation activity.