Plant chimeric binding polypeptides for universal molecular recognition
Libraries of nucleic acids encoding chimeric binding polypeptides based on plant scaffold polypeptide sequences. Also described are methods for generating the libraries.
1. A library of isolated nucleic acids encoding at least ten different polypeptides, wherein
(i) the amino acid sequence of each of the at least ten different polypeptides includes amino acids that differ from those of SEQ ID NOs:127-129 at one or more of positions 13, 14, 33, 35-36, 38, 46-47, 66, 68-69, 71, 79-80, 99, 101-102, 104, and 112-113, and the amino acid differences vary across a plurality of the polypeptides; and
(ii) the amino acid sequence of each of the at least ten different polypeptides outside of the residues corresponding to positions 13, 14, 33, 35-36, 38, 46-47, 66, 68-69, 71, 79-80, 99, 101-102, 104, and 112-113, is at least 95% identical to the sequence of SEQ ID NOs: 127-129.
2. The library of isolated nucleic acids of claim 1 , wherein the amino acid sequence of each of the at least ten different polypeptides outside of the residues corresponding to positions 13, 14, 33, 35-36, 38, 46-47, 66, 68-69, 71, 79-80, 99, 101-102, 104, and 112-113 of SEQ ID NOs: 127-129 do not vary across a plurality of the polypeptides.
3. The library of isolated nucleic acids of claim 1 , wherein the amino acid sequence of each of the at least ten different polypeptides outside of the residues corresponding to positions 13, 14, 33, 35-36, 38, 46-47, 66, 68-69, 71, 79-80, 99, 101-102, 104, and 112-113 of SEQ ID NOs: 127-129 is at least 98% identical the sequence of SEQ ID NOs: 127-129.
4. A method of generating the library of claim 1 , comprising:
(i) selecting an amino acid sequence corresponding to any one of SEQ ID NOs: 127-129 to be encoded, wherein the selected sequence differs from those of SEQ ID NOs:127-129 in at least one of variable positions 13, 14, 33, 35-36, 38, 46-47, 66, 68-69, 71, 79-80, 99, 101-102, 104, and 112-113;
(ii) chemically providing a first and a second plurality of oligonucleotides, wherein
(a) oligonucleotides of the first plurality encode amino acid subsequences of the selected amino acid sequence;
(b) oligonucleotides of the second plurality are complementary to nucleotide sequences encoding subsequences of the selected amino acid sequence; and
(c) the first and second pluralities comprise oligonucleotides have overlapping sequences complementary to one another;
(iii) combining the population of oligonucleotides to form a first mixture;
(iv) incubating the mixture under conditions effective for hybridizing the overlapping complementary sequences to form a plurality of hybridized complementary sequences; and
(v) elongating the plurality of hybridized complementary sequences to form a second mixture containing the library.