Mutations in fd phage major coat protein modulate affinity of the displayed peptide

Abstract

Multibillion-clone libraries of phages displaying guest peptides fused to the major coat protein pVIII (landscape libraries) are a rich source of probes for proteinaceous and non-proteinaceous targets. As opposed to the pIII-type fusion phages, which display peptides as independent structural domains, the guest peptides in the pVIII-fusion phages can be structurally and functionally influenced by contiguous subunits. To decipher the impact of the locale of a guest peptide on its affinity characteristics, we constructed a library of phages carrying beta-galactosidase-binding peptide ADTFAKSMQ at the N-terminus of the pVIII protein surrounded by random amino acids. It was found that mutagenesis of amino acids 12-19 (domain C) has polar effects on target binding affinity of the displayed peptide. The phages with highest affinity are characterized by: (i) a net electrostatic charge around -1 of domain C of the mutated phages at pH 7.0; (ii) a lower radius of cylinder coaxial to alpha-helix formed by domain C; (iii) a lower higher occupied molecular orbital (HOMO) of domain C leading to a decreased formation of hydrogen bonds and (iv) positively charged surface and torsion energy of domain C, which may require a conformational transition of N-terminal peptide ADTFAKSMQ for its binding with beta-galactosidase. Influence of the guest peptide on the diversity of mutations in the neighboring landscape area was also observed.

Selection of Lung Cancer-Specific Landscape Phage for Targeted Drug Delivery

“…The number of phage particles was estimated using either spectroscopy to measure the number of physical phage particles (virions) or by titering phage in a bacterial host as colony forming units (CFU). Physical titering of phage was performed when phage concentrations were higher than 10 11 virions/mL by measuring the absorbance at 269 nm and converted to vir/mL by the following formula for f8/8 and f8/9 libraries (9198 nucleotide genome) as derived previously [12]: $$1{\text{ Absorbance Unit (AU)}}_{269}=6.5\times {10}^{12}\text{virions}/\text{mL}$$…”

“…Landscape phage libraries of type f8 [10,12,13] designed on the filamentous bacteriophage vector fd-tet, consist of a collection of phage with random inserts introduced at the N-terminal end of every copy of the pVIII major coat protein of the phage vector and are subsequently expressed in ~4,000 copies across the length of the phage particle (Figure 1A). This layout dramatically alters the physical properties of the phage surface so that each phage particle is structurally unique compared to the remainder of the library and each phage possesses a specific ability to interact with the surrounding environment.…”

Phage protein‐targeted cancer nanomedicines

“…(C) Fragment of three‐dimensional structure of landscape phage showing the arrangement of pVIII units. Adapted from [107].…”

Next generation phage display by use of pVII and pIX as display scaffolds

“…This involves trimming of pIII as in CT display [52] or removing the N1-N2 cysteines of the full length pIII [53,54]. With respect to the pVIII capsid, increasing the hydrophobic packaging [55], or modifying the solvent exposed N-terminus [56], may increase display levels.…”