SMALL BINDING-PROTEINS SELECTED FROM A COMBINATORIAL REPERTOIRE OF KNOTTINS DISPLAYED ON PHAGE

Knottins are a group of small, disulphide-bonded proteins that bind wi th high specificity to their target molecules. These proteins appear t o use different faces of the protein for their interactions with diffe rent targets. Here, we attempted to create knottins with novel binding activities based on the cellulose-binding domain of the fungal enzyme cellobiohydrolase I. Variation was introduced to the face of the prot ein tl-cat binds cellulose. Seven residues, which are located in two r egions of the polypeptide chain and form a patch of about 400 Angstrom (2) on the protein surface, were simultaneously varied by random mutat ion of the gene. The repertoire was cloned for display on filamentous bacteriophage (5.5 x 10(8) clones), and selected for binding to cellul ose or to one of three enzymes (alpha-amylase, alkaline phosphatase an d beta-glucuronidase). We thereby isolated variant knottins against ce llulose (differing in sequence from the parent knottin) and also again st alkaline phosphatase. The binding to (glycosylated) alkaline phosph atase was highly specific with an affinity of about 10 mu M, required the presence of disulphide bonds and was mediated through protein (rat her than carbohydrate) contacts. Knottin scaffolds therefore appear to be a promising architecture for the creation of small folded proteins with binding activities, with the potential for improvement of bindin g affinities by mutation, or of using other faces of the protein to pr ovide greater structural diversity in the primary repertoire. (C) 1998 Academic Press Limited.