Picomolar Affinity Fibronectin Domains Engineered Utilizing Loop Length Diversity, Recursive Mutagenesis, and Loop Shuffling

Benjamin J. Hackel, Atul Kapila, K. Dane Wittrup

Research output: Contribution to journalArticlepeer-review

142 Scopus citations


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 × 107 clones and screening only 1 × 108 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.

Original languageEnglish (US)
Pages (from-to)1238-1252
Number of pages15
JournalJournal of Molecular Biology
Issue number5
StatePublished - Sep 19 2008

Bibliographical note

Funding Information:
The research was funded by CA96504, CA101830, a National Defense Science and Engineering Graduate Fellowship and a National Science Foundation Graduate Fellowship. Assistance from the MIT Flow Cytometry Core Facility is greatly appreciated. The Biophysical Instrumentation Facility for the Study of Complex Macromolecular Systems (NSF-0070319 and NIH GM68762) is gratefully acknowledged.


  • Fn3
  • affinity maturation
  • fibronectin type III domain
  • loop shuffling
  • protein engineering


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