Crystallization of a carbamatase catalytic antibody Fab fragment and its complex with a transition-state analogue.
Catalytic antibodies showing carbamatase activity have significant potential in antibody-directed prodrug therapy against tumours. The Fab fragment of an IgG1 mouse monoclonal carbamatase catalytic antibody JC1 raised against a transition-state analogue, ethyl N-(3,5-dicarboxyphenyl)-P-[N-[5'-(2",5"-dioxo-1"-pyrrolidinyl)oxy-1',5'-dioxopentyl]-4-aminophenylmethyl]phosphonamidate, was obtained by digestion of the whole antibody with papain and was purified by two-step ion-exchange chromatography. Using hanging-drop vapour-diffusion crystallization techniques, three different crystal forms of the Fab fragment were obtained in the presence and absence of the transition-state analogue. All crystals diffract X-rays to between 3.5 and 3.2 A resolution. The two crystal forms grown in the presence of the transition-state analogue contain up to four or eight copies of the Fab in the asymmetric unit and diffract to 3.5 and 3.2 A, respectively. The crystal of the Fab alone is most likely to contain only two copies of the Fab in the asymmetric unit and diffracts to beyond 3.5 A. Determination of the structure will provide insights into the active-site arrangement of this antibody and will help to increase our understanding of the molecular mechanisms by which the immune system can evolve catalytic function.
Muranova
Tatyana A
TA
Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, England.
Ruzheinikov
Sergey N
SN
Higginbottom
Adrian
A
Clipson
Jennifer A
JA
Blackburn
G Michael
GM
Wentworth
Paul
P
Datta
Anita
A
Rice
David W
DW
Partridge
Lynda J
LJ
D000595:Amino Acid Sequence; D017151:Antibodies, Catalytic / Q000737:chemistry* / Q000235:genetics; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000235:genetics; D001483:Base Sequence; D002219:Carbamates / Q000276:immunology / Q000378:metabolism; D002848:Chromatography, DEAE-Cellulose; D002852:Chromatography, Ion Exchange; D003460:Crystallization; D018360:Crystallography, X-Ray; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry* / Q000235:genetics; D007074:Immunoglobulin G / Q000737:chemistry; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA
D017151:Antibodies, Catalytic; D000911:Antibodies, Monoclonal; D002219:Carbamates; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G