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 screen this library for peptides that would bind to macromolecules of interest. Peptide diversity was achieved in an initial stock solution of peptides by using proteases under conditions in which both hydrolysis and synthesis occurred. As an example, a simple reaction containing YGG, FL and thermolysin resulted in the synthesis of YGGFL as well as many other undefined products. When low molecular weight products of a reaction containing VA, AL, and thermolysin were subsequently exposed to dipeptidase, 7 out of 9 potential dipeptides were observed. Incubation of protease with an hydrolysate of albumin and a radiolabeled peptide resulted in the radiolabel participating in reactions other than simple hydrolysis and, after 24 h, the specific activity of radiolabel was shown by high performance liquid chromatography to disperse to a level that would be necessary in the event of maximum theoretical diversity. When a binding macromolecule was exposed to this system, ligand production was amplified relative to reactions run in the absence of binding macromolecule. This protease-based peptide scrambling and binding system was utilized for the discovery of novel peptides that bind to fibrinogen.
Swann
P G
PG
Receptor Laboratories, Inc., Chicago, IL 60612, USA.
Casanova
R A
RA
Desai
A
A
Frauenhoff
M M
MM
Urbancic
M
M
Slomczynska
U
U
Hopfinger
A J
AJ
Le Breton
G C
GC
Venton
D L
DL
D002851:Chromatography, High Pressure Liquid; D010450:Endopeptidases / Q000378:metabolism*; D005340:Fibrinogen / Q000378:metabolism; D006868:Hydrolysis; D008024:Ligands; D010455:Peptides / Q000138:chemical synthesis* / Q000378:metabolism*; D011485:Protein Binding
D008024:Ligands; D010455:Peptides; D005340:Fibrinogen; D010450:Endopeptidases