Applications and characterization of peptides derived from phage-display libraries
Abstract (Summary)New developments and discoveries are presented here regarding phage-displayed peptides. First. a method was established for isolating a phage-dispiayed peptide by transferring it to the N-terminus of the maltose-binding protein (MBP) of Escherichia d i . A number of peptides were affinity-selected by an HIV- 1-specific monoclonal antibody and engineered as peptide:MBP fusions for characterization by ELISA. surface plasmon resonance, and Western blot. Analysis of peptides displayed on monomeric MBP eliminates several confounding effects on affinity caused by multivalent peptide display via the major coat protein. pVIII. Second. a new application of phage was developed for vaccine design. A conjugate in which a synthetic malarial peptide was chemically crosslinked to phage or to MBP was shown to induce a superior anti-peptide antibody response in BALBk mice. as compared to a recombinant peptide counterpart displayed either at low copy number on phage, or monovalently on MBP. The enhanced antibody response observed for the chernical conjugates was most Iikely due to a higher ratio of peptide to carrier using this approach. The level at which the synthetic peptide was coupled to phage was increased approxirnately two-fold when a reactive Lys residue was engineered ont0 the N-terminus of pVIII. The ability to conjugate synthetic peptide to phage. and to alter the phage coat by engineering pVIII, makes phage an attractive carrier for synthetic peptide-based imrnunogens. Finally. a novel structural constraint was discovered for peptides containing Cys . . . 111 residues that are displayed as N-terminal fusions to pVIII. Such peptide:pVIII hisions were found to form disulfide-rnediated homodimers on the phage coat. A nurnber of randomly-selected clones fiom libraries containing one or two tixed Cys residues. were surveyed for the presence of peptide:pVIII homodimers. Homodimerization was found to occur for al1 peptides containing a single Cys residue, whereas dimers were observed for some. but not all. peptides containing two Cys residues. A structural mode1 is presented and discussed in view of our existing knowledge of phage structure and assembly. This analysis should lead to new types of stnicturally-consttained peptide libraries.
Source Type:Master's Thesis
Date of Publication:01/01/1999