A combinatorial method for constructing libraries of long peptides displayed by filamentous phage.
We describe the construction and screening of a random peptide library displayed by filamentous phage. The peptides are expressed in multiple copies on the filamentous phage M13 as amino-terminal fusions with the major coat protein, the product of gene VIII. These libraries are efficiently screened for reactive peptides, using a combination of panning in solution followed by a plaque lift assay. Advantages of this system are that both high- and low-affinity phage clones are simultaneously identified and the analysis of non-reactive phage is minimized. The vector system utilized to construct this library enables it to be used for the construction of peptide libraries employing a combinatorial cloning strategy. This feature makes it especially suitable for construction of peptide libraries using codon-based oligonucleotide synthesis. The vectors also allow rapid optimization and modification of lead peptides by codon-based mutagenesis. A 20-amino acid long random peptide library of 1 x 10(9) members was constructed and screened for peptides that bound to (i) a monoclonal antibody recognizing the amino-terminus of beta-endorphin; (ii) a monoclonal antibody recognizing a peptide epitope derived from the v-ros oncogene product; and (iii) the constant region of murine IgG2b. The approach described here provides a means for the construction of customized libraries that can be screened with a variety of target molecules.
Haaparanta
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Ixsys Inc., San Diego, CA 92121, USA.
Huse
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D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D017104:Bacteriophage M13 / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002213:Capsid / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics; D019020:Directed Molecular Evolution / Q000379:methods*; D019040:E-Selectin / Q000235:genetics / Q000276:immunology; D005822:Genetic Vectors; D007074:Immunoglobulin G; D008722:Methods; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009856:Oncogene Proteins, Viral / Q000235:genetics / Q000276:immunology; D010452:Peptide Biosynthesis; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry / Q000235:genetics*; D020794:Receptor Protein-Tyrosine Kinases; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D001615:beta-Endorphin / Q000235:genetics / Q000276:immunology
D000911:Antibodies, Monoclonal; D004274:DNA, Recombinant; D019040:E-Selectin; D007074:Immunoglobulin G; D009856:Oncogene Proteins, Viral; D019151:Peptide Library; D010455:Peptides; D011993:Recombinant Fusion Proteins; D001615:beta-Endorphin; D020794:Receptor Protein-Tyrosine Kinases; C058682:v-ros protein, Avian sarcoma virus UR2