A single chain Fv fragment of P-glycoprotein-specific monoclonal antibody C219. Design, expression, and crystal structure at 2.4 A resolution.
A construct encoding a single chain variable fragment of the anti-P-glycoprotein monoclonal antibody C219 was made by combining the coding sequences for the heavy and light chain variable domains with a sequence encoding the flexible linker (GGGGS)3, an OmpA signal sequence, a c-myc identification tag, and a five-histidine purification tag. The construct was expressed in Escherichia coli and purified from the periplasmic fraction using a nickel chelate column and ion exchange chromatography. Three-step Western blot analysis showed that the construct retains binding affinity for P-glycoprotein. Crystals of 1.0 x 0.2 x 0.2 mm were grown in 100 mM citrate, pH 4.5, 21% polyethylene glycol 6000 in the presence of low concentrations of subtilisin, resulting in proteolytic removal of the linker and purification tags. The structure was solved to a resolution of 2.4 A with an R factor of 20.6, an Rfree of 28.5, and good stereochemistry. This result could lead to a clinically useful product based on antibody C219 for the diagnosis of P-glycoprotein-mediated multidrug resistance. The molecule will also be useful in biophysical studies of functional domains of P-glycoprotein, as well as studies of the intact molecule.
Hoedemaeker
F J
FJ
Ontario Cancer Institute and Department of Medical Biophysics, University of Toronto, Toronto M5G 2M9, Ontario, Canada.
Signorelli
T
T
Johns
K
K
Kuntz
D A
DA
Rose
D R
DR
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D000918:Antibody Specificity; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D015195:Drug Design; D004926:Escherichia coli; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000911:Antibodies, Monoclonal; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; C055966:immunoglobulin Fv