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Volume 272, Number 47, Issue of November 21, 1997 pp. 29784-29789
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

A Single Chain Fv Fragment of P-glycoprotein-specific Monoclonal Antibody C219
DESIGN, EXPRESSION, AND CRYSTAL STRUCTURE AT 2.4 Å RESOLUTION

(Received for publication, July 29, 1997, and in revised form, September 8, 1997)

From the Ontario Cancer Institute and Department of Medical Biophysics, University of Toronto, Toronto M5G 2M9, Ontario, Canada

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 × 0.2 × 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 Å with an R factor of 20.6, an R_free 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.

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