Synthesis, cloning and expression of the single-chain Fv gene of the HPr-specific monoclonal antibody, Jel42. Determination of binding constants withwild-type and mutant HPrs

The monoclonal antibody Jel42 is specific for the Escherichia coil histidin e-containing protein, HPr, which is an 85 amino acid phosphocarrier protein of the phosphoenolpyruvate:sugar phosphotransferase system. The binding do main (Fv) has been produced as a single chain Fv (scFv), The scFv gene was synthesized in vitro and coded for pelB leader peptide-heavy chain-linker-l ight chain-(His)(5) tail. The linker is three repeats from the C-terminal r epetitive sequence of eukaryotic RNA polymerase II. This linker acts as a t ag; it is the antigen for the monoclonal antibody Jel352. The codon usage w as maximized for E.coli expression, and many unique restriction endonucleas e sites were incorporated. The scFv gene incorporated into pT7-7 was highly expressed, yielding 10-30% of the cell protein as the scFv, which was foun d in inclusion bodies with the leader peptide cleaved. Jel42 scFv was purif ied by denaturation/renaturation yielding preparations with Kd values from 20 to 175 nM, However, based upon an assessment of the amount of active ref olded scFv, the binding dissociation constant was estimated to be 2.7 +/- 2 .0 nM compared with 2.8 +/- 1.6 and 3.7 +/- 0.3 nM previously determined fo r the Jel42 antibody and Fab fragment respectively, The effect of mutation of the antigen HPr on the binding constant of the scFv was very similar to the properties determined for the antibody and the Fab fragment. It was con cluded that the small percentage (similar to 6%) of refolded scFv is a true mimic of the Jel42 binding domain and that the incorrectly folded scFv can not be detected in the binding assay.