Efficient Construction of a Large Collection of Phage-Displayed Combinatorial Peptide Libraries

摘要

Selections from phage-displayed combinatorial peptide libraries are an effective strategy fornidentifying peptide ligands to target proteins. Existing protocols for constructing phage-displayed librariesnutilize either ligation into double-stranded phage DNA or Kunkel mutagenesis with single-stranded phagemidnDNA. Although the Kunkel approach rapidly provides library sizes of up to 1011, as many as 20% of thenphagemids may be non-recombinant. With several modifications to current Kunkel protocols, we havengenerated peptide libraries with sizes of up to 1011 clones and recombination frequencies approaching 100%.nThe production of phage libraries, as opposed to phagemid libraries, simplifies selection experiments byneliminating the need for helper phage. Our approach relies upon the presence of an amber stop codon in thencoding region of gene III of bacteriophage M13. Oligonucleotides containing randomized stretches of DNA arenannealed to the phage genome such that the randomized region forms a heteroduplex with the stop codon. Thenoligonucleotide is then enzymatically extended to generate covalently-closed, circular DNA, which isnelectroporated into a non-suppressor strain of Escherichia coli. If the amber stop codon is present in the DNAnmolecule, protein III is not synthesized and the phage cannot propagate itself. This method is customizable fornthe display of either random or focused peptide libraries. To date, we have constructed 22 different librariesnranging from 8-20 amino acids in length, utilizing complete or reduced codon sets.