title: Selecting soluble/foldable protein domains through single-gene or genomic ORF filtering: structure of the head domain of Burkholderia pseudomallei antigen BPSL2063.
abstract: The 1.8 Å resolution crystal structure of a conserved domain of the potential Burkholderia pseudomallei antigen and trimeric autotransporter BPSL2063 is presented as a structural vaccinology target for melioidosis vaccine development. Since BPSL2063 (1090 amino acids) hosts only one conserved domain, and the expression/purification of the full-length protein proved to be problematic, a domain-filtering library was generated using β-lactamase as a reporter gene to select further BPSL2063 domains. As a result, two domains (D1 and D2) were identified and produced in soluble form in Escherichia coli. Furthermore, as a general tool, a genomic open reading frame-filtering library from the B. pseudomallei genome was also constructed to facilitate the selection of domain boundaries from the entire ORFeome. Such an approach allowed the selection of three potential protein antigens that were also produced in soluble form. The results imply the further development of ORF-filtering methods as a tool in protein-based research to improve the selection and production of soluble proteins or domains for downstream applications such as X-ray crystallography.
authors.affiliation: Department of Biosciences, University of Milan, Via Celoria 26, 20133 Milan, Italy.
authors.forename: Louise J
authors.initials: LJ
authors.lastname: Gourlay
authors.affiliation: Institute of Biomedical Technologies, National Research Council, Via Fratelli Cervi 93, 20090 Segrate, Italy.
authors.forename: Clelia
authors.initials: C
authors.lastname: Peano
authors.affiliation: Department of Health Sciences and IRCAD, University of Eastern Piedmont, Via Solaroli 17, 28100 Novara, Italy.
authors.forename: Cecilia
authors.initials: C
authors.lastname: Deantonio
authors.affiliation: Department of Biosciences, University of Milan, Via Celoria 26, 20133 Milan, Italy.
authors.forename: Lucia
authors.initials: L
authors.lastname: Perletti
authors.affiliation: Institute of Biomedical Technologies, National Research Council, Via Fratelli Cervi 93, 20090 Segrate, Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Pietrelli
authors.affiliation: Department of Biosciences, University of Milan, Via Celoria 26, 20133 Milan, Italy.
authors.forename: Riccardo
authors.initials: R
authors.lastname: Villa
authors.affiliation: Department of Biosciences, University of Milan, Via Celoria 26, 20133 Milan, Italy.
authors.forename: Elena
authors.initials: E
authors.lastname: Matterazzo
authors.affiliation: Department of Biosciences, University of Milan, Via Celoria 26, 20133 Milan, Italy.
authors.forename: Patricia
authors.initials: P
authors.lastname: Lassaux
authors.affiliation: Department of Health Sciences and IRCAD, University of Eastern Piedmont, Via Solaroli 17, 28100 Novara, Italy.
authors.forename: Claudio
authors.initials: C
authors.lastname: Santoro
authors.affiliation: Institute of Biomedical Technologies, National Research Council, Via Fratelli Cervi 93, 20090 Segrate, Italy.
authors.forename: Simone
authors.initials: S
authors.lastname: Puccio
authors.affiliation: Department of Life Sciences, University of Trieste, Via Weiss 2, 34128 Trieste, Italy.
authors.forename: Daniele
authors.initials: D
authors.lastname: Sblattero
authors.affiliation: Department of Biosciences, University of Milan, Via Celoria 26, 20133 Milan, Italy.
authors.forename: Martino
authors.initials: M
authors.lastname: Bolognesi
mesh_terms: D000942:Antigens, Bacterial / Q000737:chemistry* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D016957:Burkholderia pseudomallei / Q000737:chemistry* / Q000235:genetics; D018360:Crystallography, X-Ray; D016680:Genome, Bacterial; D006801:Humans; D008554:Melioidosis / Q000382:microbiology*; D008958:Models, Molecular; D016366:Open Reading Frames; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D012995:Solubility
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins
keywords: Burkholderia pseudomallei; open reading frame-filtering library; protein antigen structure; soluble domain selection