Compositions for increasing polypeptide stability and activity, and related methods

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.

1 . A method of increasing thermal stability of a polypeptide comprising linking a peptide tag that has an amino acid sequence that is at least 70% identical to SEQ ID NO: 1 with a polypeptide to form a fusion polypeptide, wherein the fusion polypeptide does not have an amino acid sequence as shown in SEQ ID NO: 2. 
     
     
         2 . The method of  claim 1 , wherein the peptide tag has an amino acid sequence as shown in SEQ ID NO: 1, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, or SEQ ID NO: 18. 
     
     
         3 - 11 . (canceled) 
     
     
         12 . The method of  claim 1 , wherein the peptide tag is covalently linked to the polypeptide. 
     
     
         13 . The method of  claim 1 , wherein the peptide tag is non-covalently linked to the polypeptide. 
     
     
         14 - 15 . (canceled) 
     
     
         16 . 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. 
     
     
         17 - 20 . (canceled) 
     
     
         21 . The method of  claim 1 , wherein the polypeptide comprises a polymerase, reverse transcriptase, nuclease, pyrophosphatase, deaminase, or protease. 
     
     
         22 . The method of  claim 1 , wherein the polypeptide is a polymerase. 
     
     
         23 . The method of  claim 1 , wherein the polypeptide comprises a  Bacillus stearothermophilus  (Bst) polymerase or fragment thereof. 
     
     
         24 - 45 . (canceled) 
     
     
         46 . A fusion polypeptide comprising
 a) a peptide tag that has an amino acid sequence that is at least 70% identical to SEQ ID NO: 1; and   b) at least one polypeptide;   wherein the peptide tag is linked to the at least one polypeptide, the peptide tag increases thermal stability of the at least one polypeptide, and the fusion polypeptide does not have an amino acid sequence as shown in SEQ ID NO: 2.   
     
     
         47 . The fusion polypeptide of  claim 46 , wherein the peptide tag is covalently linked to the at least one polypeptide. 
     
     
         48 . (canceled) 
     
     
         49 . The fusion polypeptide of  claim 46 , wherein the peptide tag is linked to an amino terminus or an carboxy terminus of the at least one polypeptide. 
     
     
         50 - 75 . (canceled) 
     
     
         76 . A fusion polypeptide comprising:
 (a) a peptide tag that has an amino acid sequence that is at least 70% identical to SEQ ID NO: 1; and   (b) a polypeptide comprising a polymerase with strand displacement activity.   
     
     
         77 . The fusion polypeptide of  claim 76 , wherein the polymerase with strand displacement activity comprises  Bacillus stearothermophilus  (Bst) polymerase polypeptide. 
     
     
         78 . The fusion polypeptide of  claim 76 , wherein the polymerase with strand displacement activity is a fragment of  Bacillus stearothermophilus  (Bst) polymerase. 
     
     
         79 . The fusion polypeptide of  claim 76 , wherein the peptide tag increases thermal stability of the polypeptide comprising the polymerase with the strand displacement activity. 
     
     
         80 . The fusion polypeptide of  claim 79 , wherein the thermal stability of the polypeptide comprising the polymerase with the strand displacement activity is increased for at least 1 day at a temperature between 20° C. and 50° C. 
     
     
         81 . The fusion polypeptide of  claim 76 , wherein the peptide tag inhibits loss of protein function of the polypeptide comprising the polymerase with the strand displacement activity for at least 1 day after exposure to a temperature of at least 35° C.