title: Discovery of high-affinity peptide binders to BLyS by phage display.
abstract: B lymphocyte stimulator (BLyS) is a tumor necrosis factor (TNF) family member and a key regulator of B cell responses. We employed a phage display-based approach to identify peptides that bind BLyS with high selectivity and affinity. Sequence analysis of first-generation BLyS-binding peptides revealed two dominant peptide motifs, including one containing a conserved DxLT sequence. Selected linear peptides with this motif were found to bind BLyS with K(D) values of 1-3 microM. In order to improve the binding affinity for BLyS, consensus residues flanking the DxLT sequence were seeded into a second-generation, BLyS affinity maturation library (BAML). BAML phage were subjected to stringent binding competition conditions to select for isolates expressing high-affinity peptide ligands for BLyS. Post-selection analysis of BAML peptide sequences resulted in the identification of a core decapeptide motif (WYDPLTKLWL). Peptides containing this core motif exhibited K(D) values as low as 26 nM, approximately 100-fold lower than that of first-generation peptides. A fluorescence anisotropy assay was developed to monitor the protein-protein interaction between BLyS labeled with a ruthenium chelate, and TACI-Fc, a soluble form of a BLyS receptor. Using this assay it was found that a BAML peptide disrupts this high-affinity protein-protein interaction. This demonstrates the potential of short peptides for disruption of high affinity cytokine-receptor interactions.
authors.affiliation: Dyax Corp., 300 Technology Square, Cambridge, MA 02139, USA.
authors.forename: Tony J
authors.initials: TJ
authors.lastname: Fleming
authors.forename: Meena
authors.initials: M
authors.lastname: Sachdeva
authors.forename: Marko
authors.initials: M
authors.lastname: Delic
authors.forename: James
authors.initials: J
authors.lastname: Beltzer
authors.forename: Charles R
authors.initials: CR
authors.lastname: Wescott
authors.forename: Mary
authors.initials: M
authors.lastname: Devlin
authors.forename: Robert C
authors.initials: RC
authors.lastname: Lander
authors.forename: Andrew E
authors.initials: AE
authors.lastname: Nixon
authors.forename: Viktor
authors.initials: V
authors.lastname: Roschke
authors.forename: David M
authors.initials: DM
authors.lastname: Hilbert
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Sexton
mesh_terms: D020816:Amino Acid Motifs; D053264:B-Cell Activating Factor; D001681:Biological Assay; D017124:Conserved Sequence; D004220:Disulfides / Q000737:chemistry; D005454:Fluorescence Polarization; D006801:Humans; D008565:Membrane Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D025941:Protein Interaction Mapping / Q000379:methods; D018124:Receptors, Tumor Necrosis Factor / Q000737:chemistry* / Q000502:physiology; D012428:Ruthenium / Q000737:chemistry; D053303:Transmembrane Activator and CAML Interactor Protein; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism
chemical_list: D053264:B-Cell Activating Factor; D004220:Disulfides; D008565:Membrane Proteins; D019151:Peptide Library; D010455:Peptides; D018124:Receptors, Tumor Necrosis Factor; C108467:TNFRSF13B protein, human; C505719:TNFSF13B protein, human; D053303:Transmembrane Activator and CAML Interactor Protein; D014409:Tumor Necrosis Factor-alpha; D012428:Ruthenium