Selective enrichment and characterization of high affinity ligands from collections of random peptides on filamentous phage.
Large collections of random peptides can be expressed on the N-terminus of the pIII protein of filamentous phage and screened for binding to antibodies and other receptors. In our previous work with a monoclonal antibody (3E7) (Cwirla et al., Proc. Natl. Acad. Sci. USA 87, 6378-6382, 1990), we showed that a high proportion of the selected peptides had relatively low affinity (Kd's greater than 1 microM). Here we describe conditions for selective enrichment of phage expressing high affinity peptides. This is done by allowing the phage to interact with a low concentration of 3E7 Fab followed by extensive washing to allow dissociation of phage-bearing peptides with low affinity. These affinity selection conditions were applied to the pool of phage previously selected using a high concentration of IgG. A phage clone with the known high affinity ligand YGGFL (Kd 7.1 nM) and several other closely related peptides were isolated. The dissociation rate of 125I-3E7 Fab from several phage clones approximated that of phage expressing YGGFL. A good correlation was found between the dissociation rate of the peptides found on phage and the equilibrium binding constants of chemically synthesized peptides. The strategy of using a low concentration of receptor and extensive washing to select phage-bearing high affinity peptides, combined with assays to determine the specificity and relative affinity of peptides on isolated phage clones, should be generally applicable in using the peptides-on-phage system for discovery of high affinity receptor ligands.
Barrett
R W
RW
Department of Molecular Pharmacology, Affymax Research Institute, Palo Alto, California 94304.
Cwirla
S E
SE
Ackerman
M S
MS
Olson
A M
AM
Peters
E A
EA
Dower
W J
WJ
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism; D000915:Antibody Affinity; D002213:Capsid / Q000235:genetics; D003090:Coliphages / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005822:Genetic Vectors; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism; D008024:Ligands; D008969:Molecular Sequence Data; D010455:Peptides / Q000276:immunology / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D001615:beta-Endorphin / Q000276:immunology
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D008024:Ligands; D010455:Peptides; D011993:Recombinant Fusion Proteins; D001615:beta-Endorphin