In situ proteolysis, crystallization and preliminary X-ray diffraction analysis of a VHH that binds listeria internalin B.
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.
Department of Chemistry, California State University Fresno, 2555 E. San Ramon Avenue, Fresno, CA 93740, USA.
Ian
I
Huh
Human Health Therapeutics, National Research Council of Canada, 100 Sussex Drive, Ottawa, ON K1A 0R6, Canada.
Robert
R
Gene
Human Health Therapeutics, National Research Council of Canada, 100 Sussex Drive, Ottawa, ON K1A 0R6, Canada.
Jyothi
J
Kumaran
Human Health Therapeutics, National Research Council of Canada, 100 Sussex Drive, Ottawa, ON K1A 0R6, Canada.
C Roger
CR
MacKenzie
Department of Chemistry, California State University Fresno, 2555 E. San Ramon Avenue, Fresno, CA 93740, USA.
Cory L
CL
Brooks
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003460:Crystallization; D008087:Listeria / Q000235:genetics; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D059748:Proteolysis; D061905:Single-Domain Antibodies / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014961:X-Ray Diffraction
D001426:Bacterial Proteins; D008565:Membrane Proteins; D061905:Single-Domain Antibodies; C095768:inlB protein, Listeria monocytogenes
Listeria monocytogenes; VHH; nanobodies; single-domain antibodies; virulence factor