Expression and secretion of the S-1 subunit and C180 peptide of pertussis toxin in Escherichia coli.
The structural gene of the S-1 subunit of pertussis toxin (rS-1) and the catalytic C180 peptide of the S-1 subunit (C180 peptide) were independently subcloned downstream of the tac promoter in Escherichia coli. Both constructions included DNA encoding for the predicted leader sequence of the S-1 subunit which was inserted between the tac promoter and the structural gene. E. coli containing the plasmids encoding for rS-1 and C180 peptide produced a peptide that reacted with anti-pertussis toxin antibody and had a molecular weight corresponding to that of the cloned gene; some degradation of rS-1 was observed. Extracts of E. coli containing plasmids encoding for rS-1 and the C180 peptide possessed ADP-ribosyltransferase activity. Subcellular fractionation showed that both rS-1 and the C180 peptide were present in the periplasm, indicating that E. coli recognized the pertussis toxin peptide leader sequence. The protein sequence of the amino terminus of the C180 peptide was identical to that of authentic S-1 subunit produced by Bordetella pertussis, which showed that E. coli leader peptidase correctly processed the pertussis toxin peptide leader sequence. Two single amino acid substitutions at residue 26 (C180I-26) and residue 139 (C180S-139) which were previously shown to reduce ADP-ribosyltransferase activity were introduced into the C180 peptide. C180I-26 possessed approximately 1% of the NAD-glycohydrolase activity of the C180 peptide, suggesting that tryptophan 26 functions in the interaction of NAD with the C180 peptide. In contrast, C180S-139 possessed essentially the same level of NAD-glycohydrolase activity as the C180 peptide, suggesting that glutamic acid 139 does not function in the interaction of NAD but plays a role in a later step in the ADP-ribosyltransferase reaction.
Barbieri
J T
JT
Department of Microbiology, Medical College of Wisconsin, Milwaukee 53226.
Moloney
B K
BK
Mende-Mueller
L M
LM
D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D005796:Genes; D005798:Genes, Bacterial; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009154:Mutation; D009244:NAD+ Nucleosidase / Q000235:genetics / Q000378:metabolism; D037342:Pertussis Toxin; D010957:Plasmids; D015183:Restriction Mapping; D010566:Virulence Factors, Bordetella / Q000235:genetics* / Q000302:isolation & purification
D046911:Macromolecular Substances; D010566:Virulence Factors, Bordetella; D037342:Pertussis Toxin; D009244:NAD+ Nucleosidase