title: Cloning and expression in Escherichia coli of the gene encoding the heat-modifiable major outer membrane protein of Haemophilus influenzae type b.
abstract: One major outer membrane protein (P1) of Haemophilus influenzae type b (Hib), with an apparent molecular weight of 34,000 (34K) as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), has been shown to be heat modifiable. After heating at 100 degrees C for 5 min in 2% SDS, the P1 protein exhibits an apparent molecular weight of 49,000 (49K) in SDS-PAGE. Monoclonal antibodies (MAbs) reactive with P1 bound to the surface of Hib, and one of these MAbs had a protective effect against the development of Hib bacteremia in an animal model for invasive Hib disease. A 6-kilobase Hib DNA insert containing the gene encoding this P1 protein was cloned into Escherichia coli by using the gamma gt11 expression vector. Recombinant phage expressing P1 were identified by screening phage plaques with a MAb directed against the P1 protein. Expression of the P1 protein by an E. coli lysogen carrying the recombinant phage was independent of both vegetative phage growth and induction of lacZ gene-directed transcription of the Hib DNA insert. The Hib DNA insert encoding the P1 protein was subcloned into the plasmid vector pBR322, and a transformant containing the recombinant plasmid pFRG100 was identified with the P1 protein-directed MAb in a colony blot-radioimmunoassay. Western blot (immunoblot) analysis determined that the recombinant P1 protein possessed heat-modifiability characteristics identical to those of the native Hib protein. The P1 protein was expressed on the surface of both the E. coli lysogen containing the recombinant phage and the E. coli transformant containing pFRG100. Western blot analysis of acute- and convalescent-phase sera from infants with Hib meningitis showed that antibodies in the convalescent-phase sera recognized the P1 protein expressed by the E. coli transformant containing pFRG100. The availability of this cloned Hib DNA insert encoding the Hib P1 protein and the expression of this protein on the surface of recombinant E. coli should facilitate the investigation of P1 for both its vaccinogenic potential and its functional role in the outer membrane of Hib.
authors.affiliation: Department of Microbiology, Southwestern Graduate School of Biomedical Sciences, Dallas, Texas.
authors.forename: F R
authors.initials: FR
authors.lastname: Gonzales
authors.forename: S
authors.initials: S
authors.lastname: Leachman
authors.forename: M V
authors.initials: MV
authors.lastname: Norgard
authors.forename: J D
authors.initials: JD
authors.lastname: Radolf
authors.forename: G H
authors.initials: GH
authors.lastname: McCracken
authors.forename: C
authors.initials: C
authors.lastname: Evans
authors.forename: E J
authors.initials: EJ
authors.lastname: Hansen
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics* / Q000276:immunology; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004926:Escherichia coli; D005786:Gene Expression Regulation; D005798:Genes, Bacterial; D006192:Haemophilus Infections / Q000517:prevention & control*; D006193:Haemophilus influenzae / Q000235:genetics* / Q000276:immunology; D006358:Hot Temperature; D007116:Immunization, Passive; D008970:Molecular Weight
chemical_list: D000911:Antibodies, Monoclonal; D001425:Bacterial Outer Membrane Proteins; D004262:DNA Restriction Enzymes