A Single Chain Fv Fragment of P-glycoprotein-specific Monoclonal Antibody C219

Abstract

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.

Method for targeting protein destruction by using a ubiquitin-independent, proteasome-mediated degradation pathway

“…Protein or peptide ligands were expressed in HEK293T cells as fusion proteins with an N or C terminus appended protein known to participate in proteinubiquitination reactions, including (i) SIP, a protein known to bind the E3 ligase Siah1 and the Skp1 protein of Skp1͞cullin͞F-box protein complexes (26); (ii) Siah-1, a RING-domain containing E3 ligase (27); (iii) E7, a papillomavirus protein with reported E3 ligase activity (28,29); (iv) a fragment of the F-box protein Fbx7, in which the substrate-binding domain was substituted, leaving the Skp1-binding region; (v) ubiquitin G76V, a nonhydrolyzable variant of ubiquitin previously used to confer proteasome-sensitivity on proteins; (vi) a tandem 4Ј oligomer of ubiquitin G76V (2); and (vii) S5a, a subunit of the proteasome involved in substrate recognition (30). In most cases, the protein ligand was separated from the ubiquitinpathway domain by a FL consisting of the sequence AGGGS-(GGGGS) 3 (31). All constructs included an epitope tag, allowing verification of protein production by immunoblotting and confirmation of binding to cellular target proteins by coimmunoprecipitation assays (data not shown).…”

A role for intracellular immunization in chemosensitization of tumor cells?

“…81 The construction and the crystal structure of an anti-MDR-1 sFv were recently reported. 82 The sFv was constructed from the murine monoclonal antibody C219 that recognizes a continuous peptide epitope present in both cytoplasmic domains of PGP. Although no data were provided about the functionality of this sFv, there is no doubt that the elucidation of the crystal structure of C219 sFv will facilitate the development of sFvs with higher affinity and specificity that might be used intracellularly to inhibit PGP function.…”

Molecular Modeling and Affinity Determination of scFv Antibody: Proper Linker Peptide Enhances Its Activity

“…One approach is to use the flexible glycinerich sequences (GGGGS) 3 as tethers. 14,24 Another set of useful linkers are derived from multidomain proteins or selected by phage display technique. 1,42 There are no reasons to believe, however, that a linker suitable for one antibody will be optimal for others.…”