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 pm, the pertactin gene, encodes a polypeptide with a predicted molecular mass of 93478 Da, referred to as P.93. Expression of the pm gene in Escherichia coli leads to the synthesis of the full-length P.93 polypeptide, which is rapidly processed to the Technology and mature P.69 protein located at the cell surface. The P.93 precursor polypeptide Medicine, London is processed at both termini. A 34 amino acid long signal sequence is removed from the amino-terminus and a polypeptide sequence of about 30000 Da (P.30) is cleaved from the carboxy-terminus. Deletion of the 3' region of pm, 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 pm 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.