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Construction and characterization of a 9-mer phage display pVIII-library with regulated peptide density

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Abstract

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 PBAD 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 × 109 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.

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Correspondence to Astrid Hilde Myrset.

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Fagerlund, A., Myrset, A.H. & Kulseth, M.A. Construction and characterization of a 9-mer phage display pVIII-library with regulated peptide density. Appl Microbiol Biotechnol 80, 925–936 (2008). https://doi.org/10.1007/s00253-008-1630-z

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Keywords

  • Phage display
  • MMP-9
  • Biopanning
  • Phagemid
  • PBAD promoter