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. © 2009. Published by Oxford University Press. All rights reserved.
CITATION STYLE
Boschek, C. B., Apiyo, D. O., Soares, T. A., Engelmann, H. E., Pefaur, N. B., Straatsma, T. P., & Baird, C. L. (2009). Engineering an ultra-stable affinity reagent based on top 7. Protein Engineering, Design and Selection, 22(5), 325–332. https://doi.org/10.1093/protein/gzp007
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