NONSPECIFIC PROTEASE-CATALYZED HYDROLYSIS SYNTHESIS OF A MIXTURE OF PEPTIDES - PRODUCT DIVERSITY AND LIGAND AMPLIFICATION BY A MOLECULAR TRAP

We sought to develop a peptide library in solution and dynamically scr een this library for peptides that would bind to macromolecules of int erest. Peptide diversity was achieved in an initial stock solution of peptides by wing proteases under conditions in which both hydrolysis a nd synthesis occurred. As an example, a simple reaction containing YGG , FL, and thermolysin resulted in the synthesis of YGGFL as well as ma ny other undefined products. When low molecular weight products of a r eaction containing VA, AL, and thermolysin were subsequently exposed t o dipeptidase, 7 out of 9 potential dipeptides were observed. Incubati on of protease with an hydrolysate of albumin and a radiolabeled pepti de resulted in the radiolabel participating in reactions other than si mple hydrolysis and, after 24 h, the specific activity of radiolabel w as shown by high performance liquid chromatography to disperse to a le vel that would be necessary in the event of maximum theoretical divers ity. When a binding macromolecule was exposed to this system, ligand p roduction was amplified relative to reactions run in the absence of bi nding macromolecule. This protease-based peptide scrambling and bindin g system was utilized for the discovery of novel peptides that bind to fibrinogen. (C) 1997 John Wiley & Sons, Inc.