title: Identification, cloning, expression, and purification of Francisella lpp3: an immunogenic lipoprotein.
abstract: The severe and fatal human disease, tularemia, results from infection with the Gram-negative pathogen Francisella tularensis. Identification of surface outer membrane proteins, specifically lipoproteins, has been of interest for vaccine development and understanding the initiation of disease. We sought to identify Francisella live vaccine strain lipoproteins that could be a component of a subunit vaccine and have adjuvant properties as TLR2 agonists. We have identified a membrane lipoprotein of Francisella LVS isolated by sarkosyl extraction and gel filtration chromatography that is recognized by sera from LVS-vaccinated individuals and tularemia patients, indicating its potential diagnostic value. Sequencing of the protein by mass spectrometry indicated that it encodes the FTL_0645 open reading frame of F. holarctica LVS, which is 100% identical/homologous to FTT1416c of F. tularensis Schu S4. The predicted 137 amino acid lipoprotein encoded by FTL_0645 ORF, was expressed in Escherichia coli, purified, and demonstrated to be a lipoprotein. This recombinant lipoprotein, named Flpp3, was able to activate TLR2 and induce an immunogenic response in mice, suggesting that the E. coli-expressed Flpp3 is palmitoylated and closely resembles the native protein in structure and immunogenicity. Taken together, these data suggest that Flpp3 could be a candidate for inclusion in a F. tularensis vaccine.
authors.affiliation: Department of Microbiology, University of Washington, Seattle, WA 98195, USA; Department of Immunology, University of Washington, Seattle, WA 98195, USA. cparra@u.washington.edu
authors.forename: Maria C
authors.initials: MC
authors.lastname: Parra
authors.forename: Scott A
authors.initials: SA
authors.lastname: Shaffer
authors.forename: Adeline M
authors.initials: AM
authors.lastname: Hajjar
authors.forename: Byron M
authors.initials: BM
authors.lastname: Gallis
authors.forename: Anthony
authors.initials: A
authors.lastname: Hager
authors.forename: David R
authors.initials: DR
authors.lastname: Goodlett
authors.forename: Tina
authors.initials: T
authors.lastname: Guina
authors.forename: Sam
authors.initials: S
authors.lastname: Miller
authors.forename: Carleen M
authors.initials: CM
authors.lastname: Collins
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification*; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D005260:Female; D005604:Francisella tularensis / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D015870:Gene Expression; D006801:Humans; D008074:Lipoproteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D016415:Sequence Alignment; D014406:Tularemia / Q000276:immunology* / Q000382:microbiology
chemical_list: D001426:Bacterial Proteins; D008074:Lipoproteins; D011994:Recombinant Proteins