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Picomolar affinity fibronectin domains engineered utilizing loop length diversity, recursive mutagenesis, and loop

Benjamin J Hackel¹, Atul Kapila, K Dane Wittrup

¹Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

Journal of Molecular Biology

|July 8, 2008

Summary

Researchers developed a new method to create highly stable fibronectin type III (Fn3) domains with unprecedented affinity for lysozyme, reaching picomolar levels. This advancement enhances Fn3 domain engineering for molecular recognition applications.

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Area of Science:

Protein engineering
Biochemistry
Molecular biology

Background:

The tenth type III domain of human fibronectin (Fn3) is a versatile scaffold for molecular recognition.
Improving the selection process for stable, high-affinity Fn3 domains is crucial for biotechnological applications.

Purpose of the Study:

To enhance the robustness of selection for stable, high-affinity Fn3 domains.
To develop an efficient library design and affinity maturation scheme for Fn3 engineering.

Main Methods:

Creation of a yeast surface display library with diversified solvent-exposed loops (amino acid composition and length).
Screening using fluorescence-activated cell sorting to isolate lysozyme binders.
Affinity maturation via random mutagenesis and homologous recombination-driven loop shuffling.
Secondary affinity maturation to further optimize binding.

Main Results:

Isolated stable, monomeric Fn3 domains with 3 pM affinity for lysozyme.
Achieved a 1.1 pM binder, the highest reported affinity for an Fn3 domain.
Demonstrated high-affinity binding while maintaining protein stability and monomeric state.

Conclusions:

The developed library design and affinity maturation scheme are highly efficient for Fn3 engineering.
Loop length diversity, loop shuffling, and recursive mutagenesis are critical for achieving high-affinity binders.
This approach extends the affinity limit for the Fn3 scaffold and preserves essential protein characteristics.