Substantially pure reverse transcriptases and methods of prooduction thereof

Abstract

The present invention provides substantially pure reverse transcriptases, which are preferably substantially free from contamination with nucleic acids. The invention also provides methods for the production of these enzymes, and kits comprising these enzymes which may be used in synthesizing, amplifying or sequencing nucleic acid molecules, including through the use of the polymerase chain reaction, particularly RT-PCR.

1 . A reverse transcriptase purified by a method comprising permeabilizing a cellular source of reverse transcriptase, subjecting said permeabilized cellular source of reverse transcriptase to filtration, and isolating said reverse transcriptase, wherein said reverse transcriptase is substantially free of nucleic acids.  
     
     
         2 . The reverse transcriptase of  claim 1 , wherein said cellular source is a bacterial cell or a recombinant bacterial cell.  
     
     
         3 . The reverse transcriptase of  claim 2 , wherein said permeabilization forms spheroplasts and/or protoplasts.  
     
     
         4 . The reverse transcriptase of  claim 1 , wherein said filtration comprises microfiltration or ultrafiltration.  
     
     
         5 . The reverse transcriptase of  claim 1 , wherein said permeabilization comprises contacting said cellular source with an aqueous solution comprising a chaotropic agent or a nonionic detergent.  
     
     
         6 . The reverse transcriptase of  claim 5 , wherein said nonionic detergent is Triton X-100 or sodium deoxycholate.  
     
     
         7 . The reverse transcriptase of  claim 1 , wherein said isolating comprises column chromatography.  
     
     
         8 . The reverse transcriptase of  claim 1 , wherein said method is conducted under conditions favoring the partitioning of nucleic acids from said reverse transcriptase.  
     
     
         9 . The reverse transcriptase of  claim 8 , wherein said conditions comprise microfiltration of spheroplasts or protoplasts in the presence of ammonium sulfate.  
     
     
         10 . The reverse transcriptase of  claim 1 , wherein said reverse transcriptase is MMLV RT or MMLV RT substantially reduced in RNase H activity.  
     
     
         11 . The reverse transcriptase of  claim 5 , wherein said chaotropic agent is guanidine, urea or guanidine hydrochloride.  
     
     
         12 . A reverse transcriptase substantially free of nucleic acids.  
     
     
         13 . The reverse transcriptase of  claim 12 , wherein said reverse transcriptase is MMLV RT or MMLV H− RT.  
     
     
         14 . The reverse transcriptase of  claim 1 , wherein said reverse transcriptase is RSV H− RT, AMV H− RT, RAV H− RT, MAV RT or HIV H− RT.  
     
     
         15 . The reverse transcriptase of  claim 12 , wherein said reverse transcriptase is RSV H− RT, AMV H− RT, RAV H− RT, MAV RT or HIV H− RT.