Recombinant anti‐sialidase single‐chain variable fragment antibody

Abstract

The single-chain antibody variable fragment (scFv), with a 15-residue polypeptide linker (Gly,Ser),, of monoclonal antibody NClO 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" and 20°C. At higher protein concentrations (= 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°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 boundkialidase 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 NClO antigen-binding fragment (Fab) molecule. A complex between tern N9 sialidase and NClO 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 Fabhalidase 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 (V,) and variable light (V,) chains. A close interaction between two symmetryrelated scFv suggests that they may have crystallized as dimers.Monoclonal antibodies with their unique specificity and affinity are used widely as immunodiagnostic and therapeutic reagents [I] A scFv fragment of antibody NC10, which recognizes an epitope on influenza virus sialidase, was recently cloned and expressed in Escherichia coli [14]. The V, and V, domains were linked with the 15-residue peptide described above and also contained a hydrophilic octapeptide (FLAG) [19] at the C-terminus of the V, domain as an affinity label to aid in detection of the scFv. The purified, monomeric scFv was found to form dimers and higher-molecular-mass aggregates at a protein concentration greater than 5 mg/ml. Recently, the presence of dimers and higher-molecular-mass multimers

3D domain swapping: A mechanism for oligomer assembly

“…Singlechain Fv molecules form monomers, dimers, and higher oligomers in solution (Raag & Whitlow, 1995). The hinge loop is disordered in the structure of the anti-sialidase single chain Fv molecule, but close interactions between molecules in the crystal suggest it is a domain-swapped dimer (Kortt et al, 1994 proteins in Table 1 has an identical C-interface in the monomeric and dimeric states (Fig. 2), formed between domains linked by a hinge loop.…”

On a mouse monoclonal antibody that neutralizes all four dengue virus serotypes

“…For the mAb 9F12, this model is an obvious choice. For the scFv, the bivalent analyte model gave a better fit than a 1 : 1 Langmuir model., indicating that a large part of the scFv9F12 molecules were not monomeric (Dolezal et al, 2000;Kortt et al, 1994). Indeed, scFv9F12 elutes as a dimer in gel filtration analysis (data not shown).…”

Influenza virus neuraminidase: Structure, antibodies, and inhibitors

“…The structure has been determined and illustrates that the oligopeptide linker, a 15-mer connecting VH to VL domains, has no significant effect on the domain pairing or on the structure of the complex formed with antigen (Kortt et al, 1994).…”