Bacterial display enables efficient and quantitative peptide affinity maturation.
A quantitative screening method was developed to enable isolation and affinity maturation of peptide ligands specific for a given target from peptide libraries displayed on the outer surface of Escherichia coli using multi-parameter flow cytometry. From a large, random 15-mer peptide library, screening identified a core motif of W-E/D-W-E/D that conferred binding to vascular endothelial growth factor (VEGF). One cycle of affinity maturation resulted in the identification of several families of VEGF-binding peptides having distinct consensus sequences, from which a preferred disulfide constraint emerged. In the second affinity maturation cycle, high affinity peptides were favored by the addition of a decoy protein that bound an adjacent epitope on the display scaffold. The decoy apparently reduced rebinding or avidity effects, and the resulting peptides exhibited consensus at 12 of 19 amino acid positions. Peptides identified and affinity matured using bacterial display were remarkably similar to the best affinity matured using phage display and exhibited comparable dissociation constants (within 2-fold; K(D) = 4.7 x 10(-7) M). Screening of bacterial-displayed peptide libraries using cytometry enabled optimization of screening conditions to favor affinity and specificity and rapid clonal characterization. Bacterial display thus provides a new quantitative tool for the discovery and evolutionary optimization of protein-specific peptide ligands.
Kenrick
Sophia A
SA
Department of Chemical Engineering, University of California, Santa Barbara, CA 93106, USA.
Daugherty
Patrick S
PS
D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D008024:Ligands; D019151:Peptide Library; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism
D008024:Ligands; D019151:Peptide Library; D011994:Recombinant Proteins; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A