In situ proteolysis, crystallization and preliminary X-ray diffraction analysis of a VHH that binds listeria internalin B

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DOIResolve DOI: http://doi.org/10.1107/S2053230X1402010X
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TypeArticle
Journal titleActa Crystallographica Section F:Structural Biology Communications
ISSN2053-230X
Volume70
Pages15321535; # of pages: 4
SubjectCamelidae; Listeria; Listeria monocytogenes
AbstractThe variable region of camelid heavy-chain antibodies produces the smallest known antibody fragment with antigen-binding capability (a V<inf>H</inf>H). The V<inf>H</inf>H R303 binds internalin B (InlB), a virulence factor expressed by the pathogen Listeria monocytogenes. InlB is critical for initiation of Listeria infection, as it binds a receptor (c-Met) on epithelial cells, triggering the entry of bacteria into host cells. InlB is surface-exposed and is required for virulence, hence a V<inf>H</inf>H targeting InlB has potential applications for pathogen detection or therapeutic intervention. Here, the expression, purification, crystallization and X-ray diffraction of R303 are reported. Crystals of R303 were obtained following in situ proteolysis with trypsin. Gel filtration and SDS-PAGE revealed that trypsin removed the C-terminal tag region of R303, facilitating crystal formation. Crystals of R303 diffracted to 1.3Å resolution and belonged to the monoclinic space group P21, with unit-cell parameters a = 46.4, b = 31.2, c = 74.8Å, β = 93.8°. The crystals exhibited a Matthews coefficient of 1.95Å3Da-1 with two molecules in the asymmetric unit.
Publication date
PublisherInternational Union of Crystallography
LanguageEnglish
AffiliationNational Research Council Canada (NRC-CNRC); Human Health Therapeutics
Peer reviewedYes
NPARC number21275505
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Record identifierc8eb4b43-079d-44b2-adfd-0708f103495b
Record created2015-07-14
Record modified2016-05-09
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