Artificial antibody polypeptides

Abstract

The present invention provides a fibronectin type III (Fn3) molecule, wherein the Fn3 contains a stabilizing mutation. The present invention also provides Fn3 polypeptide monobodies, nucleic acid molecules encoding monobodies, and variegated nucleic acid libraries encoding such monobodies. Also provided are methods of preparing a Fn3 polypeptide monobody, and kits to perform the methods.

1 - 21 . (canceled) 
     
     
         22 . A method of preparing a fibronectin type III (Fn3) polypeptide monobody comprising the steps of:
 a) providing a DNA sequence encoding a plurality of Fn3 β-strand domain sequences that are linked to a plurality of loop region sequences, wherein at least one loop region contains a unique restriction enzyme site, and wherein at least one of the plurality of Fn3 β-strand domain sequences are more stable at neutral pH than wild-type Fn3;   b) cleaving the DNA sequence at the unique restriction site;   c) inserting into the restriction site a DNA segment known to encode a peptide capable of binding to a specific binding partner (SBP) or a transition state analog compound (TSAC) so as to yield a DNA molecule comprising the insertion and the DNA sequence of (a); and   d) expressing the DNA molecule so as to yield polypeptide monobody.   
     
     
         23 . A method of preparing a fibronectin type III (Fn3) polypeptide monobody comprising the steps of:
 (a) providing a replicatable DNA sequence encoding a plurality of Fn3 β-strand domain sequences that are linked to a plurality of loop region sequences, wherein the nucleotide sequence of at least one loop region is known, and wherein at least one of the plurality of Fn3 β-strand domain sequences are more stable at neutral pH than wild-type Fn3;   (b) preparing polymerase chain reaction (PCR) primers sufficiently complementary to the known loop sequence so as to be hybridizable under PCR conditions, wherein at least one of the primers contains a modified nucleic acid sequence to be inserted into the DNA;   (c) performing polymerase chain reaction using the DNA sequence of (a) and the primers of (b);   (d) annealing and extending the reaction products of (c) so as to yield a DNA product; and (e) expressing the polypeptide monobody encoded by the DNA product of (d).   
     
     
         24 . A method of preparing a fibronectin type Ill (Fn3) polypeptide monobody comprising the steps of:
 a) providing a replicatable DNA sequence encoding a plurality of Fn3 β-strand domain sequences that are linked to a plurality of loop region sequences, wherein the nucleotide sequence of at least one loop region is known, and wherein at least one of the plurality of Fn3 β-strand domain sequences are more stable at neutral pH than wild-type Fn3;   b) performing site-directed mutagenesis of at least one loop region so as to create a DNA sequence comprising an insertion mutation; and   c) expressing the polypeptide monobody encoded by the DNA sequence comprising the insertion mutation.   
     
     
         25 - 50 . (canceled)