Ja, William W. and Olsen, Brett N. and Roberts, Richard W. (2005) Epitope mapping using mRNA display and a unidirectional nested deletion library. Protein Engineering, Design and Selection, 18 (7). pp. 309-319. ISSN 1741-0126. http://resolver.caltech.edu/CaltechAUTHORS:JAWpeds05
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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.
|Additional Information:||© The Author 2005. Published by Oxford University Press. PEDS Advance Access originally published online on June 24, 2005. Received November 23, 2004; revised April 22, 2005; accepted May 18, 2005. Edited by Dario Neri. We thank Dr David S. Waugh (National Cancer Institute at Frederick) for the pDW363 in vivo biotinylation vector, Professor Pamela J. Bjorkman and Anthony M. Giannetti for time and support on the Biacore 2000, William Hunter (Biacore, Piscataway, NJ) for technical advice on SPR, Cindy I. Chen and Christopher T. Balmaseda for preparative and technical assistance with library construction and protein purification and Professor David G. Myszka (University of Utah) for generously providing the kinetic analysis software, Scrubber and CLAMP. We greatly appreciate Dr Yuri Peterson (Duke University) for suggestions on the paper. We are indebted to Dr Ian N. Hampson (St Mary’s Hospital, Manchester, UK) for valuable discussions and technical expertise on random priming and the synthesis of the fragment library. This work was supported by grants from the NIH (RO1 GM 60416) and the Beckman Foundation to R.W.R. W.W.J. was supported in part by a DOD National Defense and Engineering Graduate Fellowship.|
|Subject Keywords:||anti-polyhistidine; epitope mapping; HIS-1; in vitro selection; mRNA display; nested deletion; peptide libraries|
|Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Tony Diaz|
|Deposited On:||01 Jun 2006|
|Last Modified:||26 Dec 2012 08:53|
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