title: Expression and mutagenesis of recombinant cholera toxin A subunit.
abstract: ADP-ribosylating protein exotoxins from Vibrio cholerae (CT) and Escherichia coli (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 drastically decreases ADP ribosyltransferase activity. We have more closely defined the role of other amino acids in this region by generating modified proteins in which arginine 7 was replaced with lysine (R7K), aspartate 9 was replaced with arginine (D9R), glycine was substituted 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 proteins R7K, D9R and delta 613 exhibited undetectable ADP ribosyltransferase activity. Comparison of the tryptic digest of R7K with native CT suggested that changes in protein conformation may be responsible for the loss of ADP-ribosylation activity.
authors.affiliation: Laboratory of Microbial Ecology, National Institute of Dental Research, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: K L
authors.initials: KL
authors.lastname: Vadheim
authors.forename: Y
authors.initials: Y
authors.lastname: Singh
authors.forename: J M
authors.initials: JM
authors.lastname: Keith
mesh_terms: D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D002772:Cholera Toxin / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D004252:DNA Mutational Analysis; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis; D013329:Structure-Activity Relationship
chemical_list: D011994:Recombinant Proteins; D000246:Adenosine Diphosphate Ribose; D002772:Cholera Toxin