Purification of Q Beta replicase

Abstract

A process of producing highly pure, Q Beta replicase having a high level of activity is described. The present process allows isolation of Q Beta replicase from recombinant bacteria containing a clone of a phage DNA encoding the 65,000 weight subunit of Q Beta replicase. The present process provides an efficient method for producing pure Q Beta replicase which can readily be scaled up to commercial production levels.

I claim: 
     
       1. A method for purifying Q Beta replicase from cells in which it is produced, comprising the steps of: a) disrupting cells which produce Q Beta replicase to form a cell lysate;   b) contacting the cell lysate with polyethyleneimine under conditions appropriate for selective precipitation of nucleic acids present in the lysate, resulting in formation of a supernatant containing Q Beta replicase;   c) contacting the supernatant with a .[.anionic resin.]. .Iadd.cationic resin .Iaddend., under conditions appropriate for binding of Q Beta replicase to the resin;   d) contacting the product of step (c) with a first elution buffer under conditions appropriate for separation of Q Beta replicase from the resin;   e) contacting the product of step (d) with an .[.cationic resin.]. .Iadd.anionic resin .Iaddend.under conditions appropriate for binding of Q Beta replicase to the resin; and   f) contacting the resin of step (e) with a second elution buffer under conditions appropriate for separation of Q Beta replicase from the resin.   
     
     
       2. A method of claim 1, wherein the cells are E. coli cells which produce Q Beta replicase. 
     
     
       3. A method of claim 1, wherein cells are disrupted in step (a) by sonication or homogenization. 
     
     
       4. A method of claim 1, wherein the first elution buffer and the second elution buffer comprise salt gradients having a concentration of NaCl of from about 100 to about 400 mM. 
     
     
       5. A method of producing substantially pure Q Beta replicase, comprising the steps of: a) providing a lysate of Q Beta replicase-producing E. coli cells;   b) contacting the cell lysate with a polyethyleneimine in the presence of NaCl under conditions appropriate for selective precipitation of nucleic acids present in the lysate, resulting in formation of a supernatant containing Q Beta replicase;   c) contacting the supernatant with a .[.anionic resin.]. .Iadd.cationic resin .Iaddend.under conditions appropriate for binding of Q Beta replicase to the resin;   d) contacting the product of step (c) with a first elution buffer under conditions appropriate for separation of Q Beta replicase from the resin;   e) contacting the product of step (d) with an .[.cationic resin.]. .Iadd.anionic resin .Iaddend.under conditions appropriate for binding of Q Beta replicase to the resin; and   f) contacting the resin of step (e) with a second elution buffer under conditions appropriate for separation of Q Beta replicase from the resin.   
     
     
       6. A method of claim 5, wherein the polyethyleneimine is 10% (w/v) polyethyleneimine.