Expression of the Bordetella pertussis P.69 pertactin adhesin in Escherichia coli: fate of the carboxy-terminal domain.
The mature pertactin protein (P.69) of Bordetella pertussis can be isolated from the bacterial cell surface as a polypeptide with an apparent molecular mass of 69,000 Da as determined by sodium dodecyl sulphate gel electrophoresis. However the open reading frame of prn, the pertactin gene, encodes a polypeptide with a predicted molecular mass of 93,478 Da, referred to as P.93. Expression of the prn gene in Escherichia coli leads to the synthesis of the full-length P.93 polypeptide, which is rapidly processed to the mature P.69 protein located at the cell surface. The P.93 precursor polypeptide is processed at both termini. A 34 amino acid long signal sequence is removed from the amino-terminus and a polypeptide sequence of about 30,000 Da (P.30) is cleaved from the carboxy-terminus. Deletion of the 3' region of prn, encoding P.30, results in the expression of an intracellular form of P.69. Antiserum which recognizes P.30 was raised using synthetic peptides based on the primary amino acid sequence of the region. This anti-P.30 serum was used in a Western blot analysis of fractionated cells of B. pertussis and E. coli harbouring the intact prn gene. The P.30 polypeptide was readily detected in outer membrane fractions prepared from both of these bacterial species, although it could not be shown to be exposed at the cell surface.
Charles
I
I
Wellcome Research Laboratories, Beckenham, Kent, UK.
Fairweather
N
N
Pickard
D
D
Beesley
J
J
Anderson
R
R
Dougan
G
G
Roberts
M
M
D000595:Amino Acid Sequence; D000907:Antibodies, Bacterial; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D001886:Bordetella pertussis / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000648:ultrastructure; D015870:Gene Expression; D005798:Genes, Bacterial; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010957:Plasmids / Q000235:genetics; D011499:Protein Processing, Post-Translational; D017384:Sequence Deletion; D010566:Virulence Factors, Bordetella
D000907:Antibodies, Bacterial; D001425:Bacterial Outer Membrane Proteins; D010566:Virulence Factors, Bordetella; C068929:pertactin