A high-capacity alkaline phosphatase reporter system for the rapid analysis of specificity and relative affinity of peptides from phage-display libraries.
We describe a novel reporter enzyme cassette system which enables the analysis of large numbers of linear and cyclic peptides in terms of their binding to a specific target molecule. In this system, peptides selected for target binding from random peptide phage-display libraries are expressed as cloned fusion proteins with bacterial alkaline phosphatase. The binding specificity and relative affinity of each peptide-enzyme fusion protein is then evaluated in a target-specific ELISA. This strategy enables direct identification of the highest affinity peptides, specific for a given target, which can then be sequenced at the DNA level to derive their peptide sequences. This eliminates the need to sequence large numbers of clones to establish consensus sequences for binding peptides. This approach also eliminates the need for peptide synthesis or phage ELISA to determine relative binding affinities, which can be technically difficult. Identification of binding peptides based on specificity and relative affinity, rather than conforming to an amino acid consensus sequence, enables the rapid evaluation of hundreds of candidate peptides and identification of rare (non-consensus) binding peptides which may otherwise be missed.
Wright
R M
RM
Transplantation Research, Novartis Pharmaceuticals Corporation, 556 Morris Avenue, 07901, Summit, NJ, USA.
Dudas
D
D
Gavin
B
B
Dottavio
D
D
Hexham
J M
JM
Lake
P
P
D000469:Alkaline Phosphatase / Q000032:analysis* / Q000235:genetics; D000911:Antibodies, Monoclonal / Q000276:immunology; D002457:Cell Extracts / Q000032:analysis; D003208:Concanavalin A / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D020128:Inhibitory Concentration 50; D019151:Peptide Library; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D000911:Antibodies, Monoclonal; D002457:Cell Extracts; D019151:Peptide Library; D010455:Peptides; D011993:Recombinant Fusion Proteins; D003208:Concanavalin A; D000469:Alkaline Phosphatase