title: The Bordetella pertussis virulence factor P.69 pertactin retains its immunological properties after overproduction in Escherichia coli.
abstract: Bordetella pertussis is re-emerging in several countries with a high vaccine uptake. Analysis of clinical isolates revealed antigenic divergence between vaccine strains and circulating strains with respect to P.69 pertactin. Polymorphisms in P.69 pertactin are mainly limited to regions comprised of amino acid repeats, designated region 1 and region 2. Region 1 flanks the RGD motif involved in adherence. Although antibodies against P.69 pertactin are implicated in protective immunity, little is known about the structure and location of its epitopes. Previously we described the localization of mainly linear epitopes of both human sera and mouse monoclonal antibodies (mAbs). To study the location of conformational epitopes and to investigate the effect of variation in P.69 pertactin on vaccine efficacy, we cloned, expressed, and purified 3 naturally occurring P.69 pertactin variants, 3 mutants in which the variable regions are missing, 3 N-terminal mutants and 1 C-terminal deletion mutant. Here, we describe the procedure to clone, express, and purify up to 0.1mg P.69 pertactin and its derivatives per 1 ml Escherichia coli culture.
authors.affiliation: Laboratory for Vaccine Preventable Diseases, National Institute of Public Health and the Environment, Bilthoven, The Netherlands.
authors.forename: Marcel
authors.initials: M
authors.lastname: Hijnen
authors.forename: Pieter G M
authors.initials: PG
authors.lastname: van Gageldonk
authors.forename: Guy A M
authors.initials: GA
authors.lastname: Berbers
authors.forename: Tiest
authors.initials: T
authors.lastname: van Woerkom
authors.forename: Frits R
authors.initials: FR
authors.lastname: Mooi
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D001483:Base Sequence; D001886:Bordetella pertussis / Q000235:genetics / Q000276:immunology* / Q000472:pathogenicity*; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005798:Genes, Bacterial; D014644:Genetic Variation; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry; D010567:Pertussis Vaccine / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D017384:Sequence Deletion; D014774:Virulence / Q000235:genetics / Q000276:immunology; D010566:Virulence Factors, Bordetella / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000276:immunology*
chemical_list: D001425:Bacterial Outer Membrane Proteins; D004269:DNA, Bacterial; D010567:Pertussis Vaccine; D011994:Recombinant Proteins; D010566:Virulence Factors, Bordetella; C068929:pertactin