Journal
PROTEIN ENGINEERING DESIGN & SELECTION
Volume 18, Issue 7, Pages 309-319Publisher
OXFORD UNIV PRESS
DOI: 10.1093/protein/gzi038
Keywords
anti-polyhistidine; epitope mapping; HIS-1; in vitro selection; mRNA display; nested deletion; peptide libraries
Funding
- NIA NIH HHS [R00 AG030493] Funding Source: Medline
- NIGMS NIH HHS [R01 GM 60416, R01 GM060416-03, R01 GM060416] Funding Source: Medline
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In vitro selection targeting an anti-polyhistidine monoclonal antibody was performed using mRNA display with a random, unconstrained 27-mer peptide library. After six rounds of selection, epitope-like peptides were identified that contain two to five consecutive, internal histidines and are biased for arginine residues, without any other identifiable consensus. The epitope was further refined by constructing a high-complexity, unidirectional fragment library from the final selection pool. Selection by mRNA display minimized the dominant peptide from the original selection to a 15-residue functional sequence (peptide Cmin: RHDAGDHHHHHGVRQ; K-D = 38 nM). Other peptides recovered from the fragment library selection revealed a separate consensus motif (ARRXA) C-terminal to the histidine track. Kinetics measurements made by surface plasmon resonance, using purified Fab (antigen-binding fragment) to prevent avidity effects, demonstrate that the selected peptides bind with 10- to 75-fold higher affinities than a hexahistidine peptide. The highest affinity peptides (K-D approximate to 10 nM) encode both a short histidine track and the ARRXA motif, suggesting that the motif and other flanking residues make important contacts adjacent to the core polyhistidine-binding site and can contribute > 2.5 kcal/mol of binding free energy. The fragment library construction methodology described here is applicable to the development of high-complexity protein or cDNA expression libraries for the identification of protein-protein interaction domains.
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