Construction and screening of M13 phage libraries displaying long random peptides.
We have constructed two phage display libraries expressing N-terminal pIII fusions in M13 composed of 37 and 43 random amino acid domains, respectively. The D38 library expresses 37 random amino acids with a central alanine residue, and the DC43 library contains 43 random amino acids with a central cysteine flanked by two glycine residues, giving the displayed peptide the potential to form disulfide loops of various sizes. We demonstrate that the majority of random sequences in both libraries are compatible in pentavalent display with phage viability. The M13 phage display vector itself has been engineered to contain a factor Xa protease cleavage site to provide an alternative to acid elution during affinity selection. An in-frame amber mutation has been inserted between the pIII cloning sites to allow for efficient selection against nonrecombinant phage in the library. These libraries have been panned against mAb 7E11-C5, which recognizes the prostate-specific membrane antigen (PSM). Isolated phage display a consensus sequence that is homologous to a region in the PSM molecule.
McConnell
S J
SJ
Cytogen Corporation, Princeton, NJ 08540-5237, USA.
Uveges
A J
AJ
Fowlkes
D M
DM
Spinella
D G
DG
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D017104:Bacteriophage M13 / Q000235:genetics*; D001483:Base Sequence; D004247:DNA / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D019020:Directed Molecular Evolution / Q000379:methods*; D014644:Genetic Variation; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D019151:Peptide Library; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D004247:DNA