UNIVERSAL FIBRONECTIN TYPE III BINDING-DOMAIN LIBRARIES
First Claim
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1. A walk-through mutagenesis library of fibronectin Type 3 (FN3) domain polypeptides useful in screening for the presence of one or more polypeptides having a selected binding or enzymatic activity, said polypeptides comprising:
- (a) regions A, AB, B, C, CD, D, E, EF, F, and G having wildtype amino acid sequences of a selected native fibronectin Type 3 polypeptide, and(b) loop regions BC, DE, and FG having selected lengths, where at least one selected loop region of a selected length contains a library of walk through mutagenesis sequences expressed by a library of coding sequences that encode, at each loop position, a conserved or selected semi-conserved consensus amino acid and, if the consensus amino acid has an occurrence frequency equal to or less than a selected threshold frequency of at least 50%, a single common target amino acid and any co-produced amino acids.
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Abstract
Walk-through mutagenesis and natural-variant combinatorial fibronectin Type III (FN3) polypeptide libraries are described, along with their method of construction and use. Also disclosed are a number of high binding affinity polypeptides selected by screening the libraries against a variety of selected antigens.
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Citations
20 Claims
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1. A walk-through mutagenesis library of fibronectin Type 3 (FN3) domain polypeptides useful in screening for the presence of one or more polypeptides having a selected binding or enzymatic activity, said polypeptides comprising:
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(a) regions A, AB, B, C, CD, D, E, EF, F, and G having wildtype amino acid sequences of a selected native fibronectin Type 3 polypeptide, and (b) loop regions BC, DE, and FG having selected lengths, where at least one selected loop region of a selected length contains a library of walk through mutagenesis sequences expressed by a library of coding sequences that encode, at each loop position, a conserved or selected semi-conserved consensus amino acid and, if the consensus amino acid has an occurrence frequency equal to or less than a selected threshold frequency of at least 50%, a single common target amino acid and any co-produced amino acids. - View Dependent Claims (2, 3, 4, 5)
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6. A natural-variant combinatorial library of fibronectin Type 3 (FN3) domain polypeptides useful in screening for the presence of one or more polypeptides having a selected binding or enzymatic activity, said polypeptides comprising:
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(a) regions A, AB, B, C, CD, D, E, EF, F, and G having wildtype amino acid sequences of a selected native fibronectin Type 3 polypeptide, and (b) loop regions BC, DE, and FG having selected lengths, where at least one selected loop region of a selected length contains a library of natural-variant combinatorial sequences expressed by a library of coding sequences that encode at each loop position, a conserved or selected semi-conserved consensus amino acid and, if the consensus amino acid has a frequency of occurrence equal to or less than a selected threshold frequency of at least 50%, other natural variant amino acids, including semi-conserved amino acids and variable amino acids whose occurrence rate is above a selected minimum threshold occurrence at that position, or their chemical equivalents. - View Dependent Claims (7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)
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Specification