EXPRESSION AND CHARACTERIZATION OF RECOMBINANT SINGLE-CHAIN FV AND FVFRAGMENTS DERIVED FROM A SET OF CATALYTIC ANTIBODIES

The generation of catalytic antibodies should enable the catalysis of reactions for which no enzymatic or chemical catalyst is currently ava ilable. In previous studies, we established a series of catalytic anti bodies capable of hydrolysing p-nitrobenzyl (pNB) and p-nitrophenyl (p NP) esters. ii group of these catalytic antibodies exhibited high reac tivity and substrate specificity, yet each individual antibody demonst rated different kinetic parameters. In order to study the molecular ba sis for these differences, we have cloned, sequenced and expressed the variable regions of this group of antibodies as functional scFv and F v in bacteria. The variable region of the heavy chain is derived from a novel germline gene of the J558 family whereas the light chain comes from a germline gene previously found in our catalytic antibodies cat alysing the hydrolysis of only nitrophenyl esters, demonstrating that the heavy chain determines the specificity for the nitrobenzyl esters. Several different expression systems were examined for their ability to produce catalytically active antibodies. When expressed as an scFv, both refolded and secreted scFvs exhibited catalytic activity althoug h yields of expressed protein were low. The secreted scFvs had higher specific activity. On the other hand, Fv fragments were expressed in s ufficient quantities to allow kinetic analysis. Levels of expression w ere dependent on the sequence of V-L used. Using this expression syste m, the relative contributions of the individual light and heavy chains to catalysis and binding could be evaluated. Both original V-H and V- L regions are required for hapten binding, although the V-H is more cr ucial for catalysis. By replacing the CDR3 of the heavy chain with a r andom sequence, it was shown to be essential for both binding and cata lysis. This expression system together with site-directed mutagenesis should enable a more detailed study of the catalytic mechanism of this set of antibodies. (C) 1998 Elsevier Science Ltd. All rights reserved .