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 generating a protein scaffold binding to a specific target with a predefined binding affinity, comprising the steps of:
a) contacting a library of a scaffold-based protein derived from a stability enhanced consensus sequence of a fibronectin type III domain incorporating randomized codons wherein a polynucleotide sequence encoding a polypeptide comprising a stability enhanced consensus sequence of a fibronectin type III domain of the amino acid sequence of SEQ ID NO: 150 is provided, randomizing codons are introduced into the polynucleotide sequence at selected positions and copies of the polynucleotide sequence are propagated to form a library of polynucleotides encoding variant scaffold proteins, with the specific target; and
b) isolating a protein scaffold binding to the specific target with the predefined affinity.
2. The method of claim 1 , wherein the isolating step comprising isolating scaffold molecules binding to the specific target and testing the isolated scaffold molecules for binding affinity to the specific target.
3. The method of claim 2 , wherein the isolating step comprises panning the library with the specific target, capturing scaffold molecules bound to the specific target, and isolating the binding scaffold molecules.
4. The method of claim 2 , wherein the predefined binding affinity as measured by K D is less than or equal to about 10 −7 M.