Expression of triple-helical collagen-like products in E. coli

Patent metadata

Field	Value
Publication number	US-9382310-B2
Application number	US-201013148066-A
Country	US
Kind code	B2
Filing date	Feb 5, 2010
Priority date	Feb 6, 2009
Publication date	Jul 5, 2016
Grant date	Jul 5, 2016

Abstract

Recombinant bacterial triple-helical collagen-like proteins comprising two or more repetitive sequences of Gly-Xaa-Yaa yielding high-stability polymeric constructs without the need for post-translational modifications and which may incorporate one or more functional domains of biological or structural importance. The polymers are capable of high-yield production for a variety of applications.

First claim

We claim:

1. A recombinant bacterial collagen-like protein structure comprising a formula:
[(Gly-Xaa-Y aa) m -(insert) n ] p
wherein m is between 1 to 200, n is 1, and p is between 2 to 10,
wherein (Gly-Xaa-Yaa) m represents a tandem repeat triple helical domain wherein Xaa and Yaa are independently any natural or unnatural imino or amino acid with the proviso that neither Xaa nor Yaa is a hydroxyproline,
wherein the insert is comprised of 1 to 50 of any imino or amino acids and wherein the insert is a non-triple helical forming peptide sequence,
wherein the tandem repeat triple helical domains have a circular dichroism spectroscopy value of between 0.04 to 0.13 for the ratio of positive peak (about 220 nm) to negative peaks (about 198 nm), and
wherein the collagen-like protein structure is stable at temperatures between 35° C. and 40° C.

2. The collagen-like protein structure of claim 1 , wherein the tandem repeat triple helical domains have a proline content of greater than 19% of all residues in the Xaa and Yaa positions.

3. The collagen-like protein structure of claim 2 , wherein the tandem repeat triple helical domains have a proline content of between 19.5% and 40% of all residues in the Xaa and Yaa positions.

4. The collagen-like protein structure of claim 1 , wherein the tandem repeat triple helical domains have a concentration of charged amino acids of greater than 14% of all residues in the Xaa and Yaa positions.

5. The collagen-like protein structure of claim 1 , wherein the tandem repeat triple helical domains have a concentration of charged amino acids of between 14-35% of all residues in the Xaa and Yaa positions.

6. The collagen-like protein structure of claim 1 , further comprising a non-collagenous domain bound at either an amino terminus end or a carboxy terminus end of the collagen-like protein, which facilitates protein folding of the tandem repeat triple helical domains.

7. The collagen-like protein structure of claim 6 , wherein the non-collagenous domain is SEQ ID NO: 47.

8. The collagen-like protein structure of claim 6 , wherein the non-collagenous domain is SEQ ID NO: 47 and is bound to the protein at the amino terminus end of the collagen-like protein.

9. The recombinantly expressed protein of claim 6 , wherein the non-collagenous domain is SEQ ID NO: 51 and is bound to the protein at the carboxy terminus end of the triple helical domain.

10. The recombinantly expressed protein of claim 6 , wherein the non-collagenous domain is selected from the group consisting of a foldon, a coiled coil sequence, and a C-propeptide.

11. The recombinantly expressed protein of claim 1 , wherein the insert sequence includes at least one non-collagen natural break having a peptide sequence spaced between two glycine residues.

12. The recombinantly expressed protein of claim 11 , wherein the non-collagen natural break is selected from the group consisting of SEQ ID NOs: 12-14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 50, SEQ ID NO: 65, and combinations thereof.

13. The collagen-like protein structure of claim 1 , wherein the tandem repeat triple-helical domains aggregate at neutral pH.

14. A recombinant bacterial collagen-like protein structure comprising a formula:
[(Gly-Xaa-Y aa) m -(insert) n ] p
and at least one non-collagenous domain bound to the protein structure to at least one of an amino terminus end or a carboxy terminus end of the protein structure,
wherein m is between 1 to 200, n is 1, and p is between 2 to 10,
wherein (Gly-Xaa-Yaa) m represents a tandem repeat triple helical domain wherein Xaa and Yaa are independently any natural or unnatural imino or amino acid with the proviso that neither Xaa nor Yaa is a hydroxyproline,
wherein the insert is comprised of 1 to 50 of any imino or amino acids and wherein the insert is a non-triple helical forming peptide sequence
wherein the tandem repeat triple helical domains have a circular dichroism spectroscopy value of between 0.04 to 0.13 for the ratio of positive peak (about 220 nm) to negative peaks (about 198 nm), and
wherein the non-collagenous domain facilitates protein folding of the tandem repeat triple helical domains, and
wherein the collagen-like protein structure is stable at temperatures between 35° C. and 40° C.

15. The collagen-like protein structure of claim 14 , wherein the tandem repeat triple helical domains have a Proline content of greater than 19% of all residues in the Xaa and Yaa positions.

16. The collagen-like protein structure of claim 15 , wherein the tandem repeat triple helical domains have a Proline content of between 19.5% and 40% of all residues in the Xaa and Yaa positions.

17. The collagen-like protein structure of claim 14 , wherein the tandem repeat triple helical domains have a concentration of charged amino acids of greater than 14% of all residues in the Xaa and Yaa positions.

18. The collagen-like protein structure of claim 14 , wherein the tandem repeat triple helical domains have a concentration of charged amino acids of between 14-35% of all residues in the Xaa and Yaa positions.

19. The collagen-like protein structure of claim 18 , wherein the tandem repeat triple helical domains are stable at temperatures between 35° C. and 40° C. in its native form.

20. The collagen-like protein structure of claim 14 , wherein the non-collagenous domain is SEQ ID NO: 47.1.

21. The collagen-like protein structure of claim 14 , wherein the non-collagenous domain is SEQ ID NO: 47 and is bound to the protein at the amino terminus end of the collagen-like protein.

22. The recombinantly expressed protein of claim 14 , wherein the non-collagenous domain is SEQ ID NO: 51 and is bound to the protein at the carboxy terminus end of the triple helical domain.

23. The recombinantly expressed protein of claim 14 , wherein the non-collagenous domain is selected from the group consisting of a foldon, a coiled coil sequence, and a C-propeptide.

24. The recombinantly expressed protein of claim 14 , wherein the insert sequence includes at least one non-collagen natural break having a peptide sequence spaced between two glycine residues.

25. The recombinantly expressed protein of claim 24 , wherein the non-collagen natural break is selected from the group consisting of SEQ ID NOs: 12-14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 50, SEQ ID NO: 65, and combinations thereof.

26. The collagen-like protein structure of claim 14 , wherein the triple-helical domain aggregates at neutral pH.

27. A method of producing a recombinant collagen-like protein comprising:
(a) inserting nucleic acid sequences encoding a bacterial collagen-like protein into a single nucleic acid vector, said recombinant bacterial collagen-like protein structure comprising a formula:
[(Gly-Xaa-Y aa) m -(insert) n ] p

wherein m is between 1 to 200 n is 1 and is between 2 to 10,
wherein (Gly-Xaa-Yaa) m represents a tandem repeat triple helical domain wherein Xaa and Yaa are independently any natural or unnatural imino or amino acid with the proviso that neither Xaa nor Yaa is a hydroxyproline,
wherein the insert is comprised of 1 to 50 of any imino or amino acids and wherein the insert is a non-triple helical forming peptide sequence;
(b) optionally inserting into said vector a nucleic acid encoding a non-collagenous domain nucleic acid sequence at either or both 5′ or 3′ end of the nucleic acid encoding the

Assignees

• Mirochnitchenko Oleg
• Inouye Masayori
• Brodsky Barbara
• Ramshaw John
• Univ Rutgers

Inventors

• Mirochnitchenko Oleg
• Inouye Masayori
• Brodsky Barbara
• Ramshaw John

Classifications

• C07K2319/74

  containing a fusion for binding to a cell surface receptor · CPC title

• C07K14/315

  from Streptococcus (G), e.g. Enterococci · CPC title

• C07K14/78 Primary

  Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG] · CPC title

• C07K14/195

  from bacteria · CPC title

• C07K14/705

  Receptors; Cell surface antigens; Cell surface determinants {(tumour specific antigens C07K14/4748)} · CPC title