Journal
JOURNAL OF MOLECULAR BIOLOGY
Volume 381, Issue 5, Pages 1238-1252Publisher
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2008.06.051
Keywords
fibronectin type III domain; Fn3; protein engineering; loop shuffling; affinity maturation
Categories
Funding
- National Defense Science and Engineering Graduate Fellowship [CA96504, CA101830]
- National Science Foundation Graduate Fellowship
- Study of Complex Macromolecular Systems [NSF-0070319, NTH GM68762]
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The 10th type III domain of human fibronectin (Fn3) has been validated as an effective scaffold for molecular recognition. In the current work, it was desired to improve the robustness of selection of stable, high-affinity Fn3 domains. A yeast surface display library of Fn3 was created in which three solvent-exposed loops were diversified in terms of amino acid composition and loop length. The library was screened by fluorescence-activated cell sorting to isolate binders to lysozyme. An affinity maturation scheme was developed to rapidly and broadly diversify populations of clones by random mutagenesis as well as homologous recombination-driven shuffling of mutagenized loops. The novel library and affinity maturation scheme combined to yield stable, monomeric Fn3 domains with 3 pM affinity for lysozyme. A secondary affinity maturation identified a stable 1.1 pM binder, the highest affinity yet reported for an Fn3 domain. In addition to extension of the affinity limit for this scaffold, the results demonstrate the ability to achieve high-affinity binding while preserving stability and the monomeric state. This library design and affinity maturation scheme is highly efficient, utilizing an initial diversity of 2 x 10(7) clones and screening only 1 x 10(8) mutants (totaled over all affinity maturation libraries). Analysis of intermediate populations revealed that loop length diversity, loop shuffling, and recursive mutagenesis of diverse populations are all critical components. (c) 2008 Published by Elsevier Ltd.
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