Systematic discovery, maturation and extension of peptide binders to proteins
The invention comprises systems, methods and arrays for identification and optimization of novel peptide binders to protein targets. Embodiments include steps of peptide binder discovery, core peptide maturation, N-terminal and C-terminal extension and kinetics analysis of the final peptide binder.
1. A method of identifying a peptide binder comprising the steps of:
a. exposing a protein target of interest to an array comprising a first population of peptide binders, whereby the protein target binds to at least one peptide binder comprising the population, wherein the sequences of the peptide binders at specific locations on the array are known;
b. identifying peptide binder sequences comprising the population which bind the protein target of interest, whereby a core binder sequence is determined;
c. performing at least one alteration selected from a single amino acid substitution, a double amino acid substitution, an amino acid deletion, and an amino acid insertion of amino acids comprising the core binder sequence, whereby a second population of core binder sequences is generated;
d. exposing the second population to the protein target, whereby the protein target binds to at least one peptide sequence of the second population;
e. identifying one or more sequences of the second population demonstrating strong binding properties to the protein target, whereby a matured core binder sequence is determined;
f. performing at least one of N-terminal and C-terminal extension of the matured core binder sequence determined in step e, whereby a population of matured extended peptide binders is generated;
g. exposing the protein target of interest to an array comprising the population of matured peptide binders generated in step f; and
h. identifying peptide binder sequences of the peptides comprising the population of mature peptide binders, whereby an extended, matured core peptide binder sequence is determined.
2. The method of claim 1 , wherein steps c. and f. are transposed.
3. The method of claim 1 , wherein at least one of a label-free and affinity analysis of the extended, matured core peptide binder sequence is performed.
4. The method of claim 1 , wherein the array comprises at least one of glass, plastic, and carbon composite.
5. The method of claim 1 , wherein the peptide binders of the first population comprise the same number of amino acids.
6. The method of claim 1 , wherein the peptide binders of the first population do not include the amino acid cysteine or methionine, or histidine-proline-glutamine motifs, or amino acid repeats of 2 or more amino acids.
7. The method of claim 1 , wherein the peptide binders of the population of matured extended peptide binders include at least one of N-terminal and C-terminal wobble synthesis.
8. The method of claim 1 , wherein the core binder sequence comprises a greater number of amino acids than the number of amino acids for each of the peptides comprising the first population of peptide binders.
9. The method of claim 1 , wherein steps e. and h. comprise principled clustering analysis.
10. The method of claim 1 , wherein said steps c.-h. are repeated for the extended, matured core peptide binder sequence.
11. The method of claim 1 , wherein the protein target is an enzyme.
12. The method of claim 11 , wherein the enzyme is selected from the group consisting of a sortase, a protease, a kinase, a phosphatase, a BirA biotinylation enzyme, a ligase, a lipase, a phosphodiesterase, a collagenase, a hydrolase, and an esterase.
13. The method of claim 1 , wherein each of the peptide binders in the first population of peptide binders is selected from a 3-mer peptide, a 4-mer peptide, a 5-mer peptide, and a 6-mer peptide.
14. The method of claim 1 , wherein the each of the peptide binders in the first population of peptide binders is a 5-mer peptide.
15. The method of claim 1 , wherein the first population of peptide binders comprises a comprehensive list of all possible 5-mer peptides of a genome.
16. The method of claim 1 , wherein the first population of peptide binders comprises a comprehensive list of all possible 5-mer peptides using all natural amino acids excluding at least one of cysteine and methionine.
17. The method of claim 1 , wherein the second population of core binder sequences comprises all possible single-substitution variants of the core binder sequence.
18. The method of claim 1 , wherein specific amino acids or motifs are excluded from the first population of peptide sequences.
19. A method of identifying a peptide binder comprising the steps of:
a. exposing a protein target of interest to an array comprising a first population of peptide binders, whereby the protein target binds to at least one peptide binder comprising the population, wherein the sequences of the peptide binders at specific locations on the array are known, wherein each of the peptide binders in the first population of peptide binders is a 5-mer peptide;
b. identifying peptide binder sequences comprising the population which bind the protein target of interest, whereby a core binder sequence is determined;
c. performing at least one alteration selected from a single amino acid substitution, a double amino acid substitution, an amino acid deletion, and an amino acid insertion of amino acids comprising the core binder sequence, whereby a second population of core binder sequences is generated;
d. exposing the second population to the protein target, whereby the protein target binds to at least one peptide sequence of the second population;
e. identifying one or more sequences of the second population demonstrating strong binding properties to the protein target, whereby a matured core binder sequence is determined;
f. performing at least one of N-terminal and C-terminal extension of the matured core binder sequence determined in step e, whereby a population of matured extended peptide binders is generated;
g. exposing the protein target of interest to an array comprising the population of matured peptide binders generated in step f; and
h. identifying peptide binder sequences of the peptides comprising the population of mature peptide binders, whereby an extended, matured core peptide binder sequence is determined.