Compositions for increasing polypeptide stability and activity, and related methods

• US 9,816,078 B2
• Filed: 03/11/2016
• Issued: 11/14/2017
• Est. Priority Date: 11/19/2009
• Status: Active Grant

• Current Assignee: Solis Biodyne Ou
• Original Assignee: Solis Biodyne Ou
• Inventors: Kahre, Olev, Artma, Kadri, Kahre, Tiina
• Primary Examiner(s): Allen, Marianne P
• Application Number:
  US15/068,477
• Publication Number:
  US 20160251637A1
• Time in Patent Office: 613 Days
• Field of Search: None
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C07K 14/00 Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/21 containing a His-tag
• CPC Class Codes: C07K 2319/35 containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin
• CPC Class Codes: C07K 2319/50 containing protease site
• CPC Class Codes: C12N 9/00 Enzymes; Proenzymes; Compositions thereof preparations containing enzymes for cleaning teeth A61K8/66, A61Q11/00; medicinal preparations containing enzymes or proenzymes A61K38/43; enzyme containing detergent compositions C11D; enzymes with nucleic acid structure, e.g. ribozymes, C12N15/113; Processes for preparing, activating, inhibiting, separating or purifying enzymes preparation of malt C12C1/00
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12N 9/1276 RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase
• CPC Class Codes: C12N 9/14 Hydrolases (3)
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12N 9/50 Proteinases , e.g. Endopeptidases (3.4.21-3.4.25)
• CPC Class Codes: C12N 9/78 acting on carbon to nitrogen bonds other than peptide bonds (3.5)
• CPC Class Codes: C12N 9/96 Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
• CPC Class Codes: C12Y 207/07049 RNA-directed DNA polymerase (2.7.7.49), i.e. telomerase or reverse-transcriptase

Abstract

This disclosure provides peptides, polypeptides, fusion polypeptides, compositions, and methods for enhancing or increasing the stability of a polypeptide (e.g., Taq polymerase). Such peptides, polypeptides, fusion polypeptides, or compositions include polypeptides linked to a peptide tag that enhances the stability of the polypeptide. The peptides, polypeptides, fusion polypeptides, compositions may also enhance the activity, specificity, and/or fidelity of other polypeptides in a reaction mixture. The disclosure also provides methods of using such peptides, polypeptides, fusion polypeptides, compositions.

Claims

1. A fusion polypeptide comprising:

a peptide tag comprising an amino acid sequence that is at least 95% identical to SEQ ID NO;

1 or SEQ ID NO;

14; and

a reverse transcriptase polypeptide having reverse transcriptase activity, wherein the peptide tag is linked to the reverse transcriptase polypeptide, and the peptide tag increases thermal stability of the reverse transcriptase activity of the reverse transcriptase polypeptide.

2. The fusion polypeptide of claim 1, wherein the peptide tag has the amino acid sequence of SEQ ID NO:

14.

3. The fusion polypeptide of claim 1, wherein the peptide tag has the amino acid sequence of SEQ ID NO:

1.

4. The fusion polypeptide of claim 1, wherein the fusion polypeptide comprises the peptide tag linked to the amino-terminus of the reverse transcriptase polypeptide and further comprises a second peptide tag comprising an amino acid sequence that is at least 95% identical to SEQ ID NO:

1 or SEQ ID NO;

14, wherein the second peptide tag is linked to the carboxyl-terminus of the reverse transcriptase polypeptide.

5. The fusion polypeptide of claim 1, wherein the peptide tag increases thermal stability of the reverse transcriptase activity when the fusion polypeptide is exposed to temperatures of at least about 35°

C. for at least one week.

6. The fusion polypeptide of claim 1, wherein the peptide tag increases thermal stability of the reverse transcriptase activity when the fusion polypeptide is exposed to temperatures of at least 35°

C. for at least two weeks.

7. The fusion polypeptide of claim 1, wherein the peptide tag increases thermal stability of the reverse transcriptase activity when the fusion polypeptide is exposed to temperatures between 20°

C. and 50°

C.

8. The fusion polypeptide of claim 1, wherein the reverse transcriptase polypeptide is a Maloney Murine Leukemia Virus (M-MLV) Reverse Transcriptase.

9. The fusion polypeptide of claim 1, wherein the reverse transcriptase polypeptide comprises an RNase H activity.

10. The fusion polypeptide of claim 1, wherein the peptide tag has the amino acid sequence of SEQ ID NO:

16.

11. A method of increasing thermal stability of a reverse transcriptase polypeptide comprising:

providing a peptide tag that has an amino acid sequence with at least 95% identity to SEQ ID NO;

1 or SEQ ID NO;

14; and

linking the reverse transcriptase polypeptide to the peptide tag with at least 95% identity to SEQ ID NO;

1 or SEQ ID NO;

14.

12. The method of claim 11, wherein the peptide tag has an amino acid sequence of SEQ ID NO:

1.

13. The method of claim 11, wherein the peptide tag has an amino acid sequence of SEQ ID NO:

14.

14. The method of claim 11, wherein the peptide tag comprises a protease cleavage site or wherein the peptide tag comprises an amino acid sequence DDDDK.

15. The method of claim 11, wherein the peptide tag increases thermal stability of the reverse transcriptase polypeptide when the reverse transcriptase polypeptide is exposed to temperatures of at least about 35°

C. for at least one week.

16. The method of claim 11, wherein the peptide tag increases thermal stability of the reverse transcriptase polypeptide when the reverse transcriptase polypeptide is exposed to temperatures of at least about 35°

C. for at least two weeks.

17. The method of claim 11, wherein the peptide tag increases thermal stability of the reverse transcriptase polypeptide when the reverse transcriptase polypeptide is exposed to temperatures between 20°

C. and 50°

C.

18. The method of claim 11, wherein the reverse transcriptase is a Maloney Murine Leukemia Virus (M-MLV) Reverse Transcriptase.

19. The method of claim 11, wherein the reverse transcriptase polypeptide comprises an RNase H activity.

20. The method of claim 11, wherein the peptide tag has an amino acid sequence of SEQ ID NO:

16.