Construction of high-complexity combinatorial phage display peptide libraries

Random peptide libraries displayed on the surface of filamentous bacterioph age are widely used as tools for the discovery of ligands for biologically relevant macromolecules, including antibodies, enzymes, and cell surface re ceptors. Phage display results in linkage of an affinity-selectable functio n (the displayed peptide) to the DNA encoding that function, allowing selec tion of individual binding clones by interative cycles of in vitro panning and in vivo amplification. Critical to the success of a panning experiment is the complexity of the library: the greater the diversity of clones withi n the library, the more likely the library contains sequences that will bin d a given target with useful affinity. A method for construction of high-co mplexity (greater than or equal to 10(9) independent clones) random peptide libraries is presented. The key steps are highly efficient binary ligation under conditions where the vector is relatively dilute, with only a modest molar excess of insert, followed by efficient electrotransformation into E scherichia coil. Library design strategies and a protocol for rapid sequenc e characterization are also presented, (C) 2001 Academic Press.