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, the amino acid sequence of each polypeptide comprising:
C 1 -X 1 -C 2 -X 2 -C 3 -X 3 -C 4 , wherein
(i) subsequence C 1 is selected from SEQ ID NOs:1-30, subsequence C 2 is selected from SEQ ID NOs:31-60, subsequence C 3 is selected from SEQ ID NOs:61-90; subsequence C 4 is selected from SEQ ID NOs:91-120, and each of C 1 -C 4 comprise up to 10 single amino acid substitutions, deletions, insertions, or additions to the selected subsequence;
(ii) C 1 -C 4 are homogeneous across a plurality of the encoded polypeptides
(iii) each of X 1 -X 3 is an independently variable subsequence consisting of 2-20 amino acids; and
(iv) each of X 1 -X 3 are heterogeneous across a plurality of the encoded polypeptides.
2. A method of generating the library of claim 1 , comprising:
(i) providing a parental nucleic acid encoding a parental polypeptide comprising the amino acid sequence: C 1 -X 1 -C 2 -X 2 -C 3 -X 3 -C 4 , wherein subsequence C 1 is selected from SEQ ID NOs:1-30, subsequence C 2 is selected from SEQ ID NOs:31-60, subsequence C 3 is selected from SEQ ID NOs:61-90; subsequence C 4 is selected from SEQ ID NOs:91-120; each of C 1 -C 4 comprises up to 10 single amino acid substitutions, deletions, insertions, or additions to the selected subsequence; and each of X 1 -X 3 is an independent subsequence consisting of 2-20 amino acids;
(ii) replicating the parental nucleic acid under conditions that introduce up to 10 single amino acid substitutions, deletions, insertions, or additions to the X 1 , X 2 , or X 3 subsequences, whereby a population of randomly varied subsequences encoding X 1 ′, X 2 ′, or X 3 ′ is generated; and
(iii) the population of randomly varied subsequences X 1 ', X 2 ′, or X 3 ′ is substituted, into a population of parental nucleic acids at the positions corresponding to those that encode X 1 , X 2 , or X 3 .
3. A method of generating the library of claim 1 , comprising:
(i) selecting an amino acid sequence comprising the amino acid sequence C 1 -X 1 -C 2 -X 2 -C 3 -X 3 -C 4 to be encoded, wherein
(a) subsequence C 1 is selected from SEQ ID NOs:1-30, subsequence C 2 is selected from SEQ ID NOs:31-60, subsequence C 3 is selected from SEQ ID NOs:61-90, and subsequence C 4 is selected from SEQ ID NOs:91-120;
(b) each of C 1 -C 4 comprises up to 10 single amino acid substitutions, deletions, insertions, or additions to the selected subsequence;
(c) each of X 1 , X 2 , and X 3 consists of an amino acid sequence 2-20 amino acids in length;
(ii) providing a first plurality and a second plurality of oligonucleotides, wherein
(a) oligonucleotides of the first plurality encode the C 1 -C 4 subsequences and multiple heterogeneous X 1 -X 3 variant subsequences X 1 ′-X 3 ′;
(b) oligonucleotides of the second plurality are complementary to nucleotide sequences encoding the C 1 -C 4 subsequences and to nucleotide sequences encoding multiple heterogeneous X 1 ′-X 3 ′ subsequences; and
(c) the oligonucleotides of the first and second pluralities 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.