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Protein Science (2007), 16:476-484. Published by Cold Spring Harbor Laboratory Press. Copyright © 2007 The Protein Society
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Design, expression, and stability of a diverse protein library based on the human fibronectin type III domain

C. Anders Olson1 and Richard W. Roberts2

1 Biochemistry and Molecular Biophysics Option, California Institute of Technology, Pasadena, California 91125, USA
2 Departments of Chemistry and Chemical Engineering, University of Southern California, Los Angeles, California 90089, USA

(RECEIVED August 14, 2006; FINAL REVISION December 16, 2006; ACCEPTED December 18, 2006)

Protein libraries based on natural scaffolds enable the generation of novel molecular tools and potential therapeutics by directed evolution. Here, we report the design and construction of a high complexity library (30 x 1013 sequences) based on the 10th fibronectin type III domain of human fibronectin (10FnIII). We examined the bacterial expression characteristics and stability of this library using a green fluorescent protein (GFP)-reporter screen, SDS-PAGE analysis, and chemical denaturation, respectively. The high throughput GFP reporter screen demonstrates that a large fraction of our library expresses significant levels of soluble protein in bacteria. However, SDS-PAGE analysis of expression cultures indicates the ratio of soluble to insoluble protein expressed varies greatly for randomly chosen library members. We also tested the stabilities of several representative variants by guanidinium chloride denaturation. All variants tested displayed cooperative unfolding transitions similar to wild-type, and two exhibited free energies of unfolding equal to wild-type 10FnIII. This work demonstrates the utility of GFP-based screening as a tool for analysis of high-complexity protein libraries. Our results indicate that a vast amount of protein sequence space surrounding the 10FnIII scaffold is accessible for the generation of novel functions by directed as well as natural evolution.

Keywords: 10FnIII; combinatorial protein library; in vitro selection; GFP reporter screen


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Copyright © 2007 by The Protein Society.