Re-Engineering The Bacteriophage Coat for Avid Display of Fibronectin Domains

Abstract

Phage display is a compelling technique of synthetic binding protein engineering which allows for a firm genotype-phenotype linkage which can be applied to selection and evolution of engineered binding protein scaffolds. Mutations can be made to the pVIII protein providing capability to significantly increase the surface display of large proteins on a phage particle 1 . Random and rational mutagenesis of the major coat protein, pVIII, was performed on the first 30 residues in 10-mer segments. The array of mutations imbedded into the wild type pVIII was mass produced to construct a phage display library. Promising mutants will selected by the screening of avid activity by ELISA selection. The effective clones were paired with DNA from the fibronectin Gr2 library, and captured on ELISA plates by binding interaction with hen egg lysozyme for quantification by plate reader. Initial experiments yielded no avid clones, but this is likely due to the weak affinity of the first Fn chosen for capture ELISA in these experiments. Adjustments were made and are being tested to discover pVIII clones with avid functionality.