Conversion of an antibody into an enzyme which cleaves the protein HPr

Jel 42 is an IgG which binds to the small bacterial protein, HPr and the st ructure of the complex is known at high resolution. The IgG was expressed a s a single chain variable fragment (scFv) and the binding to HPr was assess ed by fluorescence polarization of fluorescein-labelled HPr. The binding co nstant for the Ige was about 20-fold higher than the scFv. Inspection of th e structure of the complex suggested that it might be possible to convert t he scFv into a bond-specific protease by the introduction of three catalyti c residues; a glutamate to increase the nucleophilicity of a nearby water m olecule, a lysine to increase the polarizability of the carbonyl group and a histidine to provide a proton to convert the amine into a better leaving group. By trial and error it was found that a fourth residue had to be conv erted into glycine in order to maintain the integrity of complimentarity-de termining region three of the heavy chain (CDRH3) at the binding interface. The resulting quadruple mutant still bound to HPr and unlike other mutants , showed weak protease activity as judged from the fluorescence polarizatio n assay. The activity was maximum at pH 6 consistent with a requirement for a protonated histidine residue. With the aid of HPr fluorescein-labelled a t two different positions, it was demonstrated that the size of the product s was consistent with cleavage occurring in the vicinity of the target pept ide bond. The activity was specific for HPr since an excess of bovine serum albumin did not interfere with the reaction. (C) 1999 Elsevier Science Ltd . All rights reserved.