Construction and characterization of a 9-mer phage display pVIII-library with regulated peptide density.
The construction of a new phagemid vector for display of peptides on the pVIII major coat protein of filamentous bacteriophage is described, in which expression of pVIII-peptide fusions was placed under the control of the arabinose-inducible P(BAD) promoter. The new phagemid showed excellent capacity for the regulation of peptide expression, as judged by enzyme-linked immunosorbent assay (ELISA) and electron microscopy of immunogold-labeled FLAG peptides displayed on phages. Regulation of the density of peptide fusions displayed on phages may offer advantages in the search for new peptide ligands due to the possibility of regulating the stringency of binding, reducing selection based on avidity effects during biopanning. Furthermore, the peptide expression in the absence of inducer was effectively shut off, minimizing growth bias of individual clones. A 9-mer phage display library prepared using the constructed phagemid was generated by insertion of randomly synthesized oligonucleotides close to the N-terminal of the pVIII protein. The library comprised a total of 9.4 x 10(9) unique transformants, and was confirmed to show high diversity. The functional utility of the library was confirmed by the successful affinity selection of peptides binding to matrix metalloproteinase-9 (MMP-9). The majority of selected peptides shared the consensus motif R(D/N)XXG(M/L)(V/I)XQ, not previously selected during biopanning against MMP-9.
Fagerlund
Annette
A
GE Healthcare Medical Diagnostics, FoUII-3, Nydalen, 0401 Oslo, Norway.
Myrset
Astrid Hilde
AH
Kulseth
Mari Ann
MA
D000595:Amino Acid Sequence; D001089:Arabinose / Q000235:genetics / Q000378:metabolism; D036022:Capsid Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D017912:Inovirus / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009842:Oligopeptides; D019151:Peptide Library; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D036022:Capsid Proteins; D009842:Oligopeptides; D019151:Peptide Library; D010455:Peptides; D011993:Recombinant Fusion Proteins; C085706:FLAG peptide; D001089:Arabinose; D020780:Matrix Metalloproteinase 9