Compositions for increasing polypeptide stability and activity, and related methods
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.
1. A method of increasing the thermal stability of a polypeptide comprising covalently linking the polypeptide at the amino-terminus to a peptide tag to form a fusion polypeptide, wherein:
(a) the peptide tag is at least 95% identical to SEQ ID NO: 1; and
(b) the fusion polypeptide does not have the amino acid sequence of SEQ ID NO: 2.
2. The method of claim 1 , wherein the peptide tag has an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 16.
3. The method of claim 1 , wherein the peptide tag is encoded by a nucleic acid sequence of SEQ ID NO: 3 or SEQ ID NO: 17.
4. The method of claim 1 , wherein the peptide tag comprises at least three histidine residues.
5. The method of claim 1 , wherein the polypeptide is erythropoietin, human Leukemia Inhibitory Factor (hLIF), granulocyte macrophage colony-stimulating factor (GM-CSF), insulin, vascular endothelial growth factor (VEGF), leptin, or bevacizumab.
6. The method of claim 1 , wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 22.
7. The method of claim 1 , wherein the polypeptide is a thermostable protein or enzyme.
8. The method of claim 7 , wherein the enzyme is a polymerase, reverse transcriptase, nuclease, pyrophosphatase, deaminase, or protease.
9. The method of claim 8 , wherein the polymerase is a DNA polymerase I, Thermus aquaticus DNA polymerase I (Taq), Thermococcus gorgonarius DNA polymerase (Tgo), Thermus thermophilics (Tth) DNA polymerase, or ZO5 DNA polymerase.
10. The method of claim 8 , wherein the pyrophosphatase is a Thermoplasma acidophilum pyrophosphatase (TAPP).
11. The method of claim 8 , wherein the deaminase is a Pyrococcus horikoshii dCTP deaminase.
12. The method of claim 7 , wherein the thermostable protein is a cosmetic peptide or polypeptide.
13. The method of claim 1 , wherein the polypeptide comprises at least two polypeptides.
14. The method of claim 13 , wherein the polypeptide comprises an enzyme and a double strand binding protein.
15. The method of claim 14 , wherein the double strand binding protein comprises an amino acid sequence of SEQ ID NO: 10.
16. The method of claim 1 , wherein the peptide tag is at least 98% identical to SEQ ID NO:1.
17. The method of claim 1 , wherein covalently linking the polypeptide causes the polypeptide to have thermal stability at 35° C.
18. The method of claim 1 , wherein covalently linking the polypeptide causes the polypeptide to have thermal stability at −20° C.