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PEDS Advance Access published online on March 25, 2009
Protein Engineering Design and Selection, doi:10.1093/protein/gzp007
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© The Author 2009. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Engineering an ultra-stable affinity reagent based on Top7

Curt B. Boschek1, David O. Apiyo1,3, Thereza A. Soares2, Heather E. Engelmann1, Noah B. Pefaur1,4, Tjerk P. Straatsma2 and Cheryl L. Baird1,5

1Cell Biology and Biochemistry Group 2Computational Biology and Bioinformatics Group, Pacific Northwest National Laboratory, PO Box 999, MS K4-12, Richland, WA 99352, USA

5 To whom correspondence should be addressed. E-mail: cheryl.baird{at}pnl.gov

Antibodies are widely used for diagnostic and therapeutic applications because of their sensitive and specific recognition of a wide range of targets; however, their application is limited by their structural complexity. More demanding applications require greater stability than can be achieved by immunoglobulin-based reagents. Highly stable, protein-based affinity reagents are being investigated for this role with the goal of identifying a suitable scaffold that can attain specificity and sensitivity similar to that of antibodies while performing under conditions where antibodies fail. We have engineered Top7—a highly stable, computationally designed protein—to specifically bind human CD4 by inserting a peptide sequence derived from a CD4-specific antibody. Molecular dynamics simulations were used to evaluate the structural effect of the peptide insertion at a specific site within Top7 and suggest that this Top7 variant retains conformational stability over 100°C. This engineered protein specifically binds CD4 and, consistent with simulations, is extremely resistant to thermal and chemical denaturation—retaining its secondary structure up to at least 95°C and requiring 6 M guanidine to completely unfold. This CD4-specific protein demonstrates the functionality of Top7 as a viable scaffold for use as a general affinity reagent which could serve as a robust and inexpensive alternative to antibodies.

Keywords: engineering affinity/molecular dynamics simulations/protein scaffold/protein stability/Top7

Received May 29, 2008; revised October 15, 2008; accepted October 26, 2008.

3 Present address: Beckman Coulter, Immunoassay Business Center, Chaska, MN 55318, USA.

4 Present address: Department of Protein Biochemistry, ZymoGenetics, Seattle, WA 98102, USA.


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