title: Cloning and sequencing of Haemophilus influenzae outer membrane protein P6.
abstract: P6, a 16,600-dalton protein present in the outer membranes of both typeable and nontypeable strains, may be an important antigen in immunity to Haemophilus influenzae. The gene encoding P6 of a nontypeable strain of H. influenzae was cloned by using bacteriophage lambda gt11. Four recombinant phages were detected by screening plaques with monoclonal antibodies and a polyclonal antiserum. One recombinant phage, clone O, produced a full-length gene product which was expressed at a high yield. The DNA insert contained within this phage was cloned into the plasmid vector pUC18 to create the recombinant plasmid pBUD1. An Escherichia coli transformant containing this plasmid produced a protein which had an apparent molecular weight identical to that of H. influenzae P6, as determined by Western blot (immunoblot) analyses. Expression of the P6 polypeptide by both clone 0 and the transformant was independent of induction of the lac operon by isopropyl-beta-D-thiogalactopyranoside, suggesting that transcription was from the promoter of the P6 gene. Immunoelectron microscopy using a monoclonal antibody with specificity for a P6 surface epitope detected the presence of P6 on the surface of the transformant. The insert in pBUD1 was cut down in size to approximately 800 base pairs. The resultant plasmid, pBUD5, also coded for a full-length gene product. DNA sequence analysis revealed that the P6 gene contains transcriptional and translational sequences resembling those recognized in E. coli and a signal sequence characteristic of procaryotic membrane proteins. In addition, the carboxy terminus of this signal sequence shares homology with a common sequence found in bacterial lipoproteins, suggesting that P6 is a lipoprotein. Posttranslational proteolytic cleavage of the signal sequence would result in a protein composed of 134 amino acids.
authors.affiliation: Department of Medicine, University of New York, Buffalo 14215.
authors.forename: M B
authors.initials: MB
authors.lastname: Nelson
authors.forename: M A
authors.initials: MA
authors.lastname: Apicella
authors.forename: T F
authors.initials: TF
authors.lastname: Murphy
authors.forename: H
authors.initials: H
authors.lastname: Vankeulen
authors.forename: L D
authors.initials: LD
authors.lastname: Spotila
authors.forename: D
authors.initials: D
authors.lastname: Rekosh
mesh_terms: D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D005798:Genes, Bacterial; D006193:Haemophilus influenzae / Q000235:genetics*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics; D014169:Transformation, Bacterial
chemical_list: D001425:Bacterial Outer Membrane Proteins; D004269:DNA, Bacterial; D011994:Recombinant Proteins