4 research outputs found
Selecting soluble/foldable protein domains through single-gene or genomic ORF filtering: structure of the head domain of Burkholderia pseudomallei antigen BPSL2063
The 1.8\u2005\uc5 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 \u3b2-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
Designing Probes for Immunodiagnostics: Structural Insights into an Epitope Targeting <i>Burkholderia</i> Infections
Structure-based epitope
prediction drives the design of diagnostic
peptidic probes to reveal specific antibodies elicited in response
to infections. We previously identified a highly immunoreactive epitope
from the peptidoglycan-associated lipoprotein (Pal) antigen from <i>Burkholderia pseudomallei</i>, which could also diagnose <i>Burkholderia cepacia</i> infections. Here, considering the high
phylogenetic conservation within <i>Burkholderia</i> species,
we ask whether cross-reactivity can be reciprocally displayed by the
synthetic epitope from <i>B. cenocepacia</i>. We perform
comparative analyses of the conformational preferences and diagnostic
performances of the corresponding epitopes from the two <i>Burkholderia</i> species when presented in the context of the full-length proteins
or as isolated peptides. The effects of conformation on the diagnostic
potential and cross-reactivity of Pal peptide epitopes are rationalized
on the basis of the 1.8 Ă… crystal structure of <i>B. cenocepacia</i> Pal and through computational analyses. Our results are discussed
in the context of designing new diagnostic molecules for the early
detection of infectious diseases