Roles of the disulfide bond and the carboxy-terminal region of the S1 subunit in the assembly and biosynthesis of pertussis toxin.
A Bordetella pertussis expression system was developed to analyze the structure-function relationship, in vivo assembly, and biosynthesis of pertussis toxin. The toxin structural gene was first deleted from the B. pertussis chromosome; into the resulting B. pertussis strain the toxin gene was introduced on a low-copy-number, broad-host-range plasmid. The amount of pertussis toxin produced and secreted with this expression system was in the same order of magnitude as that produced by B. pertussis Tohama I, indicating that although the plasmid may be present in more than one copy per cell, overproduction of the toxin was not achieved in B. pertussis. Expression of mutant pertussis toxin genes in which the codon for Cys-41 was deleted or altered or in which the carboxy-terminal region was deleted showed that both the single intrachain disulfide bond and the carboxy-terminal region of S1 are essential for the stable expression, assembly, and secretion of S1. On the other hand, the B oligomer was efficiently secreted in the culture medium in the absence of the S1 subunit. The secreted B oligomer contained S2, S3, and S4 subunits as evidenced by enzyme-linked immunosorbent assay and was fully functional with respect to haptoglobin binding. Furthermore, the deletion of the hydrophobic carboxy-terminal region has a drastic effect on S1 subunit solubility; however, inclusion of the hydrophobic region was not sufficient for assembly and secretion, indicating that other interactions involving amino acids beyond residue 207 of the S1 subunit are also required.
Antoine
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R
Department of Molecular and Cellular Biology, Smith Kline-Biologicals, Rixensart, Belgium.
Locht
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C
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D001886:Bordetella pertussis / Q000235:genetics; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D004220:Disulfides; D005798:Genes, Bacterial; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D037342:Pertussis Toxin; D011487:Protein Conformation; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship; D010566:Virulence Factors, Bordetella / Q000096:biosynthesis*
D004220:Disulfides; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D010566:Virulence Factors, Bordetella; D037342:Pertussis Toxin