Small binding proteins selected from a combinatorial repertoire of knottins displayed on phage.
Knottins are a group of small, disulphide-bonded proteins that bind with high specificity to their target molecules. These proteins appear to use different faces of the protein for their interactions with different 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 protein that binds cellulose. Seven residues, which are located in two regions of the polypeptide chain and form a patch of about 400 A2 on the protein surface, were simultaneously varied by random mutation of the gene. The repertoire was cloned for display on filamentous bacteriophage (5.5 x 10(8) clones), and selected for binding to cellulose or to one of three enzymes (alpha-amylase, alkaline phosphatase and beta-glucuronidase). We thereby isolated variant knottins against cellulose (differing in sequence from the parent knottin) and also against alkaline phosphatase. The binding to (glycosylated) alkaline phosphatase was highly specific with an affinity of about 10 microM, required the presence of disulphide bonds and was mediated through protein (rather 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 binding affinities by mutation, or of using other faces of the protein to provide greater structural diversity in the primary repertoire.
Smith
G P
GP
MRC Centre for Protein Engineering, Cambridge CB2 2QH, UK.
Patel
S U
SU
Windass
J D
JD
Thornton
J M
JM
Winter
G
G
Griffiths
A D
AD
D000469:Alkaline Phosphatase / Q000378:metabolism; D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000235:genetics*; D001483:Base Sequence; D002480:Cellulase / Q000378:metabolism; D002482:Cellulose / Q000378:metabolism; D043366:Cellulose 1,4-beta-Cellobiosidase; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D014242:Trichoderma / Q000201:enzymology
D017931:DNA Primers; D010455:Peptides; D002482:Cellulose; D000469:Alkaline Phosphatase; D002480:Cellulase; D043366:Cellulose 1,4-beta-Cellobiosidase