title: Purification and characterization of outer membrane protein P6, a vaccine antigen of non-typeable Haemophilus influenzae.
abstract: Outer membrane protein P6 is a promising vaccine antigen with potential to prevent infections caused by non-typeable Haemophilus influenzae. A convenient and reliable method for the purification of P6 and an assessment of the purity, yield, protein structure, antigenicity and immunogenicity of the purified protein are described. The method begins with intact H. influenzae and utilizes a series of incubations and centrifugations using a single buffer to remove all cell components with the exception of the peptidoglycan to which the P6 is associated. P6 is dissociated from the complex with heat and the insoluble peptidoglycan is removed by centrifugation. The procedure yields highly purified P6. Contamination with lipooligosaccharide is less than 0.025 endotoxin U per microgr P6. The yield of P6 is approximately 2 mg of P6 per l H. influenzae culture. The purified P6 retains both the secondary and tertiary structure as measured by circular dichroism and analysis with monoclonal antibodies. The purified P6 is immunogenic in animals. A convenient method for purifying P6 which retains antigenicity and immunogenicity will be an important tool for future studies of the vaccine potential of P6.
authors.affiliation: Department of Microbiology, State University of New York at Buffalo, Buffalo, NY, USA.
authors.forename: R J
authors.initials: RJ
authors.lastname: Karalus
authors.forename: T F
authors.initials: TF
authors.lastname: Murphy
mesh_terms: D000328:Adult; D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D002942:Circular Dichroism; D018073:Haemophilus Vaccines / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D006193:Haemophilus influenzae / Q000737:chemistry* / Q000276:immunology; D006801:Humans; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013183:Sputum / Q000382:microbiology
chemical_list: D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D018073:Haemophilus Vaccines; C069990:P6 outer membrane protein, Haemophilus