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 analy sis of large numbers of linear and cyclic peptides in terms of their bindin g to a specific target molecule. In this system, peptides selected for targ et binding from random peptide phage-display libraries are expressed as clo ned fusion proteins with bacterial alkaline phosphatase. The binding specif icity and relative affinity of each peptide-enzyme fusion protein is then e valuated in a target-specific ELISA, This strategy enables direct identific ation of the highest affinity peptides, specific for a given target, which can then be sequenced at the DNA level to derive their peptide sequences. T his eliminates the need to sequence large numbers of clones to establish co nsensus sequences for binding peptides. This approach also eliminates the n eed for peptide synthesis or phage ELISA to determine relative binding affi nities, which can be technically difficult. Identification of binding pepti des based on specificity and relative affinity, rather than conforming to a n amino acid consensus sequence, enables the rapid evaluation of hundreds o f candidate peptides and identification of rare (non-consensus) binding pep tides which may otherwise be missed. (C) 2001 Published by Elsevier Science B.V.