Stabilized fibronectin domain compositions, methods and uses
A protein scaffold based on a consensus sequence of fibronectin type III (FN3) proteins, such as the tenth FN3 repeat from human fibronectin (human Tenascin), including isolated nucleic acids that encode a protein scaffold, vectors, host cells, and methods of making and using thereof, exhibit enhanced thermal and chemical stability while presenting six modifiable loop domains which can be engineered to form a binding partner capable of binding to a target for applications in diagnostic and/or therapeutic compositions, methods and devices.
1. A method of constructing a library of a scaffold-based protein derived from a stability enhanced consensus sequence of a fibronectin type III domain incorporating randomized codons in order to produce polypeptide variants, comprising the steps of:
providing a polynucleotide sequence encoding a polypeptide comprising a stability enhanced consensus sequence of a fibronectin type III domain selected from the group consisting of SEQ ID NOs: 142-151;
introducing randomizing codons into the polynucleotide sequence at selected positions; and
propagating copies of the polynucleotide sequence to form a library of polynucleotides encoding variant scaffold proteins.
2. The method of claim 1 , wherein the randomizing codons are selected from the group NNS and NNK.
3. The method of claim 2 , wherein the randomizing codons are incorporated at positions encoding at least one loop region selected from the group consisting of residues at or about positions 13-16, 22-28, 38-43, 51-54, 60-64, and 75-81 of SEQ ID NO: 142-151.
4. A library produced by the method of claim 3 .