Recombinant anti-sialidase single-chain variable fragment antibody. Characterization, formation of dimer and higher-molecular-mass multimers and the solution of the crystal structure of the single-chain variable fragment/sialidase complex

Abstract

The single-chain antibody variable fragment (scFv), with a 15-residue polypeptide linker (Gly4Ser)3, of monoclonal antibody NC10 was expressed in Escherichia coli and purified to homogeneity. This scFv molecule, refolded from 6 M guanidine hydrochloride, was predominantly a monomer of 27 kDa and was stable on storage at 4 degrees and 20 degrees C. At higher protein concentrations (approximately 5 mg/ml) dimer and higher-molecular-mass multimers were formed and freezing enhanced this aggregation. The dimer was not stable and dissociated to monomer at 20 degrees C with a half-life of approximately 8 days. The higher-molecular-mass multimers and dimer dissociated to monomer in 60% ethylene glycol. Both the monomer and dimer were active and with tern N9 sialidase yielded complexes of 276 kDa and 569 kDa, respectively, indicating that four scFv molecules bound/sialidase tetramer and that the dimer was bivalent and cross-linked two sialidase tetramers. Binding studies at low concentrations and using radiolabelled scFv indicated that the binding affinity of the dimer was approximately twofold higher than that of the monomer, and the binding affinities of the scFv were similar to that of the parent NC10 antigen-binding fragment (Fab) molecule. A complex between tern N9 sialidase and NC10 scFv was crystallized and the structure of the complex was solved at 0.3-nm resolution by X-ray diffraction. Comparison of this scFv/sialidase structure with the parent Fab/sialidase structure revealed that the modes of attachment of scFv and Fab to sialidase were very similar. There was no discernible electron density for the peptide linker joining the variable heavy (VH) and variable light (VL) chains. A close interaction between two symmetry-related scFv suggests that they may have crystallized as dimers.