The fibronectin type III domain as a scaffold for novel binding proteins

The fibronectin type III domain (FN3) is a small autonomous folding unit wh ich occurs in many animal proteins involving in ligand binding. The beta-sa ndwich structure of FN3 closely resembles that of immunoglobulin domains. W e have prepared a phage display library of FN3 in which residues in two sur face loops were randomized. We have selected mutant FN3s which bind to a te st ligand, ubiquitin, with significant affinities, while the wild-type FN3 shows no measurable affinity. A dominant clone was expressed as a soluble p rotein and its properties were investigated in detail. Heteronuclear NMR ch aracterization revealed that the selected mutant protein retains the global fold of FN3. It also has a modest conformational stability despite mutatio ns at 12 out of 94 residues. These results clearly show the potential of FN 3 as a scaffold for engineering novel binding proteins. (C) 1998 Academic P ress.