TRANSMEMBRANE BETA BARREL PROTEINS

Abstract

The present disclosure provides non-naturally occurring beta barrel proteins as defined, self-complementing multipartite beta barrel proteins, uses of such proteins, and methods for designing such proteins.

Description

Claims

1. A non-naturally occurring beta barrel protein comprising the formula X1-Z1-X2-Z2-X3-Z3-X4-Z4-X5-Z5-X6-Z6-X7-Z7-X8-Z8, wherein: X1 comprises at least two amino acid residues, wherein the C-terminal residue in X1 is G;Z1 is a beta strand consisting of 10 amino acid residues, wherein residue 1 is S, T or D, residue 9 is G and residue 10 is W or Y, and wherein residues 2, 4, 6, and 8 are hydrophobic residues or G;X2 is a loop comprising at least 5 amino acids;Z2 is a beta strand consisting of 12 amino acid residues, wherein residues 5 and 6 are G, residue 9 is Y, residue 12 is S, T, or D or wherein residue 12 is S or T, and residues 1, 3, 7, and 11 are hydrophobic residues or G;X3 is a beta turn consisting of two amino acids in length;Z3 is a beta strand consisting of 9 amino acid residues, wherein residues 6 and 8 are G, residues 7 and 9 are W or Y, and residues 1, 3 and 5 are hydrophobic residues or G;X4 is a loop comprising at least 5 amino acids;Z4 is a beta strand consisting of 14 amino acid residues, wherein residue 1 is N or Q, residues 6-8 are G, residue 11 is Y, residue 14 is S, T, or D or wherein residue 14 is S or T, and residues 3, 5, 9, and 13 are hydrophobic residues or G;X5 is a beta turn consisting of two amino acids in length;Z5 is a beta strand consisting of 11 amino acid residues, wherein residue 3 is P, residue 8 is G, residue 11 is Y or W, and residues 1, 5, 7, and 9 are hydrophobic residues or G;X6 is a loop comprising at least 5 amino acids;Z6 is a beta strand consisting of 14 amino acid residues, wherein residue 3 is P, residues 6 and 8 are G, residue 11 is Y, residue 14 is S, T, or D or wherein residue 14 is S or T, and residues 1, 5, 7, 9, and 13 are hydrophobic residues or G;X7 is a beta turn consisting of two amino acids in length;Z7 is a beta strand consisting of 9 amino acid residues, wherein residue 8 is G, residues 7 and 9 is W or Y, and residues 1, 3, and 5 are hydrophobic residues or G;X8 is a loop comprising at least 5 amino acids;Z8 is a beta strand consisting of 12 amino acid residues, wherein residue 1 is N or Q, residue 6 is G, residue 9 is Y, and residues 1, 3, 5, 7, and 11 are hydrophobic residues or G.

2. The protein of claim 1, wherein the C-terminal residues in X1 are PG or QG.

3. (canceled)

4. The protein of claim 1, wherein residue 1 in Z1 is S or T.

5. The protein of claim 1, wherein none of X2, X4, X6, or X8 comprise consecutively the amino acid residues across a single row of Table 1.

6. The protein of claim 1, wherein X3, X5, and X7 independently have P, E, or D at residue 1; and N, G, E, D, Q, or Y at position 2.

7. The protein of claim 1, wherein Z1 residue 5 is Y, Z5 residue 4 is Y, or both.

8. The protein of claim 1, wherein X2, X4, X6, or X8 each independently comprise an amino acid sequence selected from the group consisting of the amino acid sequence of SEQ ID NOS:22-26.

9. The protein of claim 1, wherein residue 2 of X2 is Y.

10. The protein of claim 1, wherein one or more of the following is true: Z1 residue 8 is A;Z3 residue 5 is A;Z5 residue 7 is A;Z6 residue 5 and residue 7 are A or G; and/orZ8 residue 5 is A or G.

11. The protein of claim 1, wherein one or both of the following is true: Z3 residue 4 is E or D and Z1 residue 5 is Y; and/orZ7 residue 6 is E or D and Z5 residue 4 is Y.

12. The protein of claim 1, wherein one or more of X1, X2, X4, X6, and X8 comprise an added functional domain.

13. (canceled)

14. The protein of claim 1, comprising the amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence selected from SEQ ID NOS:1-19, wherein residues in parentheses are optional and may be present or absent.

15. (canceled)

16. The protein of claim 1, comprising the amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence selected from SEQ ID NOS:20-21.

17. A protein comprising the amino acid sequence at least 50%, identical to the amino acid sequence selected from SEQ ID NOS:1-21, wherein residues in parentheses are optional and may be present or absent.

18. (canceled)

19. A non-naturally occurring, self-complementing multipartite beta barrel protein, comprising at least a first polypeptide component and a second polypeptide component, wherein the at least first polypeptide component and the second polypeptide component are not covalently linked, wherein in total the at least first polypeptide component and the second polypeptide component comprise domains X1-Z1-X2-Z2-X3-Z3-X4-Z4-X5-Z5-X6-Z6-X7-Z7-X8-Z8, wherein each domain is as defined in claim 1; wherein (a) each beta strand is fully present within one polypeptide component of the at least first polypeptide component and the second polypeptide component, (b) none of the at least first polypeptide component and the second polypeptide component include each of Z1, Z2, Z3, Z4, Z5, Z6, Z7, and Z8; and (c) one of domains X2, X4, X6, and X8 may be partially or wholly absent in each of the first polypeptide and the second polypeptide.

20. A nucleic acid encoding the beta barrel protein of claim 1.

21. An expression vector comprising the nucleic acid of claim 20 operatively linked to a control sequence.

22. A recombinant host cell comprising the expression vector of claim 21.

23. A pharmaceutical composition, comprising (a) the beta barrel protein of claim 1; and(b) a pharmaceutically acceptable carrier.

24. Method for using the beta barrel protein of claim 1 for scaffolding binding epitopes and functional domains on liposomes, cell surface, or detergent micelles, for drug delivery, or as ion, water or small-molecule permeable transmembrane channels.

25. (canceled)