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

Autor: Huh, Ian, Gene, Robert, Kumaran, Jyothi, MacKenzie, C. Roger, Brooks, Cory L.
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Zdroj: Acta Crystallographica: Section F, Structural Biology Communications; Nov2014, Vol. 70 Issue 11, p1532-1535, 4p
Abstrakt: The variable region of camelid heavy-chain antibodies produces the smallest known antibody fragment with antigen-binding capability (a VHH). The VHH 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 VHH 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 1.3 Å resolution and belonged to the monoclinic space group P21, with unit-cell parameters a = 46.4, b = 31.2, c = 74.8 Å, β = 93.8°. The crystals exhibited a Matthews coefficient of 1.95 Å3 Da−1 with two molecules in the asymmetric unit. [ABSTRACT FROM AUTHOR]
Databáze: Complementary Index