title: Stable expression of meningococcal class 1 protein in an antigenically reactive form in outer membranes of Escherichia coli.
abstract: The entire gene encoding the class 1 outer membrane protein of Neisseria meningitidis is located on a 2.2kb fragment, obtained on digestion of chromosomal DNA with Xbal. This Xbal fragment from strain MC50 (subtype P1-16), which had previously been cloned in bacteriophage M13, has been transferred to the plasmid vector pMTL20. The resulting plasmid (pPORA100) was propagated in Escherichia coli (JM109) and cell lysates were subjected to SDS-PAGE. Western blotting with anti-class 1 protein antibodies revealed constitutive expression of a protein of 41 kD, corresponding to the class 1 protein of the parent meningococcal strain, which was absent in the E. coli control. Fractionation of E. coli cells carrying the recombinant plasmid revealed that the protein was exclusively located in the outer membrane, and N-terminal amino acid analysis of the expressed protein revealed that normal processing of the signal peptide had occurred. Immuno-gold electron microscopy showed that the protective epitope recognized by a P1-16 subtype-specific monoclonal antibody was exposed in an antigenically reactive form on the surface of E. coli cells carrying plasmid pPORA100. In contrast, expression in E. coli of a second plasmid (pPORA104) lacking the coding sequence for the first 15 amino acids of the signal peptide resulted in accumulation of recombinant class 1 protein only in the cytoplasm of the cells. Thus the presence of the meningococcal signal sequence ensures expression of this meningococcal porin protein in an antigenically native conformation in outer membranes of E. coli, while its absence results in expression of a soluble protein.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Microbiology, University of Southampton Medical School, Southampton General Hospital, UK.
authors.forename: D A
authors.initials: DA
authors.lastname: White
authors.forename: A K
authors.initials: AK
authors.lastname: Barlow
authors.forename: I N
authors.initials: IN
authors.lastname: Clarke
authors.forename: J E
authors.initials: JE
authors.lastname: Heckels
mesh_terms: D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000235:genetics; D001425:Bacterial Outer Membrane Proteins / Q000096:biosynthesis / Q000235:genetics*; D001483:Base Sequence; D002462:Cell Membrane / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics* / Q000276:immunology; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D009345:Neisseria meningitidis / Q000235:genetics*; D010957:Plasmids; D018272:Porins; D011401:Promoter Regions, Genetic; D011995:Recombination, Genetic
chemical_list: D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D018272:Porins; C057699:porin protein, Neisseria