Protein Engineering, Vol. 13, No. 8, 565-574,
August 2000
© 2000 Oxford University Press
ScFv multimers of the anti-neuraminidase antibody NC10: shortening of the linker in single-chain Fv fragment assembled in VL to VH orientation drives the formation of dimers, trimers, tetramers and higher molecular mass multimers
CSIRO Health Sciences and Nutrition and CRC for Diagnostic Technologies, 343 Royal Parade, Parkville, Victoria, 2 Biomolecular Research Institute, 343 Royal Parade, Parkville, Victoria, Australia 3052 and 3 Department of Haematology, Cambridge University, Hills Road, Cambridge CB2 2XY, UK
Synthetic genes encoding single-chain variable fragments (scFvs) of NC10 anti-neuraminidase antibody were constructed by joining the VL and VH domains with linkers of fifteen, five, four, three, two, one and zero residues. These VL–VH constructs were expressed in Escherichia coli and the resulting proteins were characterized and compared with the previously characterized NC10 scFv proteins assembled in VH–VL orientation. Size-exclusion chromatography and electron microscope images of complexes formed between various NC10 scFvs and anti-idiotype Fab' were used to analyse the oligomeric status of these scFvs. The result showed that as the linker length between VL and VH was reduced, different patterns of oligomerization were observed compared with those with VH–VL isomers. As was the case for VH–VL orientation, the scFv-15 VL–VH protein existed mainly as a monomer whereas dimer (diabody) was a predominant conformation for the scFv-5, scFv-4 and scFv-3 VL–VH proteins. In contrast to the VH–VL isomer, direct ligation of VL to VH led to the formation of predominantly a tetramer (tetrabody) rather than to an expected trimer (triabody). Furthermore, the transition between dimers and higher order oligomers was not as distinct as for VH–VL. Thus reducing the linker length in VL–VH from three to two residues did not precisely dictate a transition between dimers and tetramers. Instead, two-residue as well as one-residue linked scFvs formed a mixture of dimers, trimers and tetramers.
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