Method for cloning and expression of Rhodothermus obamensis DNA polymerase I large fragment in E. coli

Abstract

The present invention provides a novel thermostable DNA polymerase I obtainable from Rhodothermus obamensis , which possesses 3′-5′ exonuclease activity and has a half-life of about 35 minutes at 94° C. This polymerase also contains a tyrosine residue in the ribosome binding site which improves incorporation of dideoxyribonucleic acids. Also provided are isolated DNA and vectors encoding this polymerase, as well as its large fragment, and methods for producing recombinant enzyme using the same.

What is claimed is:  
     
         1 . A substantially pure thermostable DNA polymerase I obtainable from  Rhodothermus obamensis  (JCM 9785).  
     
     
         2 . The substantially pure thermostable DNA polymerase I of  claim 1 , wherein said polymerase possesses 3′-5′ exonuclease activity and has a half-life of about 35 minutes at 94° C.  
     
     
         3 . The substantially pure thermostable DNA polymerase I of  claim 2 , wherein said polymerase has a molecular weight of about 104 kDa.  
     
     
         4 . The substantially pure thermostable DNA polymerase I of  claim 2 , wherein said polymerase is encoded by a DNA segment comprising the DNA sequence of SEQ ID NO: 1 or functional equivalents thereof.  
     
     
         5 . An isolated DNA segment encoding the thermostable DNA polymerase I of  claim 1 .  
     
     
         6 . The isolated DNA segment of  claim 6 , wherein said DNA segment comprises the DNA sequence of SEQ ID NO: 1 or functional equivalents thereof.  
     
     
         7 . An isolated DNA segment encoding the large fragment of  Rhodothermus obamensis  (JCM 9785) DNA polymerase I.  
     
     
         8 . The isolated DNA segment of  claim 7 , wherein said DNA segment comprises the DNA sequence of SEQ ID NO: 3 or functional equivalents thereof.  
     
     
         9 . A recombinant vector comprising the isolated DNA of any one of claims  5 - 8 .  
     
     
         10 . The recombinant vector of  claim 9 , wherein said vector comprises pAII17-Rob polI large fragment, pLysS.  
     
     
         11 . A host cell transformed with the recombinant vector of claims  9  or  10 .  
     
     
         12 . The transformed host cell of  claim 11 , wherein said transformed host cell comprises  E. coli  ER2566[pAII17-Rob polI large fragment, pLysS] (ATCC No. ______).  
     
     
         13 . A method for producing recombinant  R.obamensis  DNA polymerase I, said method comprising culturing the transformed host cell of  claim 11  under conditions suitable to allow the expression of said recombinant  R. obamensis  DNA polymerase I and recovering recombinant  R. obamensis  DNA polymerase I.  
     
     
         14 . A recombinant  R. obamensis  DNA polymerase I produced by the method of  claim 13 .  
     
     
         15 . A recombinant  R. obamensis  DNA polymerase I large fragment, wherein said polymerase I large fragment has a molecular weight of about 71 kDa, possesses 3′-5′ exonuclease activity and has a half-life of about 35 minutes at 94° C.  
     
     
         16 . A DNA polymerase I composition comprising the recombinant  R. obamensis  DNA polymerase I large fragment of  claim 15  and an approximately 60 kDa  E. coli  GroEL protein, wherein the thermostability of said polymerase I large fragment is increased by the presence of said GroEL protein.