Journal of Molecular Biology
Volume 284, Issue 4, 11 December 1998, Pages 1141-1151
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Regular article
The fibronectin type III domain as a scaffold for novel binding proteins1

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Abstract

The fibronectin type III domain (FN3) is a small autonomous folding unit which occurs in many animal proteins involving in ligand binding. The β-sandwich structure of FN3 closely resembles that of immunoglobulin domains. We have prepared a phage display library of FN3 in which residues in two surface loops were randomized. We have selected mutant FN3s which bind to a test ligand, ubiquitin, with significant affinities, while the wild-type FN3 shows no measurable affinity. A dominant clone was expressed as a soluble protein and its properties were investigated in detail. Heteronuclear NMR characterization revealed that the selected mutant protein retains the global fold of FN3. It also has a modest conformational stability despite mutations at 12 out of 94 residues. These results clearly show the potential of FN3 as a scaffold for engineering novel binding proteins.

Keywords

molecular recognition
combinatorial libraries
phage display
protein engineering

Abbreviations

BSA
bovine serum albumin
CDR
complementarity determining region
FN3
fibronectin type III domain
GuHCl
guanidine hydrochloride
HSQC
heteronuclear single quantum correlation
NOESY
nuclear Overhauser effect spectroscopy
TBS
Tris buffered saline
TOCSY
total correlation spectroscopy
VH
the variable domain of the immunoglobulin heavy chain
VL
the variable domain of the immunoglobulin light chain
cfu
colony forming units

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Edited by J. Wells