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High-affinity fragment complementation of a fibronectin type III domain and its application to stability enhancement

¹ Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, Illinois 60637, USA
² Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, USA

(RECEIVED May 25, 2005; FINAL REVISION July 28, 2005; ACCEPTED August 15, 2005)

The tenth fibronectin type III (FN3) domain of human fibronectin (FNfn10), a prototype of the ubiquitous FN3 domain, is a small, monomeric -sandwich protein. In this study, we have bisected FNfn10 in each loop to generate a total of six fragment pairs. We found that fragment pairs bisected at multiple loops of FNfn10 show complementation in vivo as tested with a yeast two-hybrid system. The dissociation constant of these fragment pairs determined in vitro were as low as 3 nM, resulting in one of the tightest fragment complementation systems reported so far. Furthermore, we show that the affinity of fragment complementation is correlated with the stability of the uncut parent protein. Exploring this correlation, we screened a yeast two-hybrid library of one fragment and identified mutations that suppress the effect of a destabilizing mutation in the other fragment. One of the identified mutations significantly increased the stability of the uncut wild-type protein, proving that fragment complementation can be used as a novel strategy for the selection of proteins with enhanced stability.

Keywords: protein engineering; reconstitution; library screening; -sheet; surface loops

Abbreviations: CI2, chymotrypsin inhibitor 2 • FN3, fibronectin type III domain • FNfn10, tenth FN3 of human fibronectin • GuHCl, guanidine hydrochloride • HSQC, heteronuclear single-quantum correlation • NMR, nuclear magnetic resonance • PCR, polymerase chain reaction.

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051603005.

Reprint requests to: Shohei Koide, Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637, USA; e-mail: skoide{at}uchicago.edu; fax: (773) 702-0439.

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