Chimeric proteins
This invention relates to modular proteins that interact with one or more target molecules. The chimeric proteins comprise two or more repeat domains, such as tetratricopeptide repeat domains; inter-repeat loops linking the repeat domains; and one or more peptide ligands. Each peptide ligand is located in an inter-repeat loop or at the N or C terminus of the chimeric protein. The peptide ligands may include heterologous peptidyl binding motifs, such as short linear motifs (SLiMs). Chimeric proteins with various configurations and methods for their production and use are provided.
1. A method of producing a chimeric protein comprising:
providing a first nucleic acid encoding first and second repeat domains linked by inter-repeat loops; and
combining said first nucleic acid, a second nucleic acid encoding a first binding domain that binds to a first target molecule and a third nucleic acid encoding a second binding domain that binds to a second target molecule to generate a chimeric nucleic acid encoding a chimeric protein comprising said first and said second repeat domains linked by inter-repeat loops and said first and second binding domains, where said first and said second binding domains are, independently, located in an inter-repeat loop or comprise the N and/or C terminus of the chimeric protein; and
expressing said chimeric nucleic acid to produce the chimeric protein.
2. The method of claim 1 , wherein said chimeric protein comprises helix-turn-helix repeat domains.
3. The method of claim 2 , wherein said chimeric protein comprises tetratricopeptide (TPR) repeat domains.
4. The method of claim 3 , wherein a TPR repeat domain-comprises the amino acid sequence Y-X 1 X 2 X 3 X 4 ;
wherein Y is an amino acid sequence of a repeat domain selected from the group of sequences presented in Tables 4 to 6 or a variant thereof and X 1 , X 2 , X 3 , X 4 are independently any amino acid.
5. The method of claim 4 , wherein Y—X 1 X 2 X 3 X 4 comprises the amino acid sequence;
AEAWYNLGNAYYKQGDYQKAIEYYQKALEL-X 1 X 2 X 3 X 4 ; or
AEALNNLGNVYREQGDYQKAIEYYQKALEL-X 1 X 2 X 3 X 4 ; or
AEAWYNLGNAYYKQGDYQKAIEYYQKALEL-X 1 X 2 X 3 X 4 ; or
AEALNNLGNVYREQGDYQRAIEYYQRALEL-X 1 X 2 X 3 X 4 ; or
AEALRNLGRVYRRQGRYQRAIEYYRRALEL-X 1 X 2 X 3 X 4 ;
wherein X 1 , X 2 , X 3 , X 4 are independently any amino acid, and optionally wherein X 1 is D and/or wherein X 2 is P.
6. The method of claim 1 , wherein said chimeric protein comprises 2-5 repeat domains.
7. The method of claim 1 , wherein said first and said second binding domains are independently located in first and second inter-repeat loops.
8. The method of claim 1 , wherein a binding domain comprises the N terminus or a binding domain comprising the C terminus or wherein said first and second binding domains independently comprise the N and C termini.
9. The method of claim 8 , wherein the binding domain comprising the N-terminus comprises an α helix, the binding domain comprising the C-terminus comprises an α helix the binding domains comprising the N and C termini comprise an α helix.
10. The method of claim 9 , wherein the binding domain comprising the N-terminus comprises the sequence X n -XYXXXIXXYXXXLXX-X 1 X 2 XX, where residues denoted by X are independently any amino acid, X 1 and X 2 are independently any amino acid and n is 0 or any number, and optionally wherein X 1 is D and/or wherein X 2 is P.
11. The method of claim 2 , wherein the binding domain comprising the C-terminus comprises the sequence X 1 X 2 XX-XXAXXXLXX[AV]XXXXX-X n , where residues denoted by X are independently any amino acid, X 1 , and X 2 are independently any amino acid n is 0 or any number, and optionally wherein X 1 is D and/or wherein X 2 is P.
12. The method of claim 1 , wherein said first binding domain of said chimeric protein binds a target molecule selected from the group consisting of: β-catenin, KRAS, tankyrase, c-myc, n-myc, ras, notch and aurora A, α-synuclein, β-amyloid, tau, superoxide dismutase, huntingtin, oncogenic histone deacetylase, and oncogenic histone methyltransferase.
13. The method of claim 1 , wherein said second binding domain of said chimeric protein binds an E3 ubiquitin ligase.
14. The method of claim 13 , wherein said chimeric protein comprises
(i) an N terminal peptide ligand that binds a target protein and a C terminal peptide ligand that binds an E3 ubiquitin ligase, or
(ii) an inter-repeat peptide ligand that binds a target protein and a C terminal peptide ligand that binds an E3 ubiquitin ligase, or
(iii) an inter-repeat peptide ligand that binds a target protein and an N terminal peptide ligand that binds an E3 ubiquitin ligase, or
(iv) a C terminal domain that binds a target protein and an N terminal peptide ligand that binds an E1 ubiquitin ligase, or
(v) an inter-repeat binding domain that binds an E3 ubiquitin ligase and an N terminal binding domain that binds a target protein, or
(vi) an inter-repeat binding domain that binds an E3 ubiquitin ligase and a C terminal binding.
15. The method of claim 1 , wherein said chimeric protein further comprises a linker linking an inter-repeat loop to a binding domain.
16. The method of claim 1 , wherein each of said first and said second binding domain of said chimeric protein is, independently, non-hydrophobic.