Method for purifying protein and glucose dehydrogenase

Abstract

A method of purifying a target protein from a solution, in which the target protein containing an electron transfer protein is dissolved, with the use of liquid chromatography. The liquid chromatography is performed by introducing the above-described protein solution into a tank filled with a packing agent, thus bonding the target protein to the packing agent, removing impurities, and then eluting the target protein from the packing agent with the use of an eluent containing a hydroxycholanoic acid salt. As an example of the above protein, glucose dehydrogenase containing a protein having an activity of dehydrogenating glucose is cited. The liquid chromatography is performed by combining hydrophobic chromatography with anion exchange chromatography.

1 . A method for purifying a target protein from a protein solution containing the target protein by using liquid chromatography, wherein 
 the liquid chromatography comprises:    a first step of introducing the protein solution into a column filled with a packing agent and causing the packing agent to hold the target protein; and    a second step of eluting the target protein by using an eluent containing a hydroxy-cholate.    
   
   
       2 . The method for purifying protein according to  claim 1 , wherein the target protein contains an electron transfer protein.  
   
   
       3 . The method for purifying protein according to  claim 2 , wherein the target protein is provided by a glucose dehydrogenase containing a protein having glucose dehydrogenation activity.  
   
   
       4 . The method for purifying protein according to  claim 1 , wherein the packing agent is provided by an ion-exchange gel.  
   
   
       5 . The method for purifying protein according to  claim 4 , wherein the ion-exchange gel contains a quaternary ammonium group as an ion-exchange group.  
   
   
       6 . The method for purifying protein according to  claim 3 , wherein the electron transfer protein has a molecule weight of approximately 43 kDa in SDS-gel electrophoresis under a reducing environment, 
 the protein which has glucose dehydrogenation activity having a molecule weight of approximately 60 kDa in SDS-gel electrophoresis under a reducing environment.    
   
   
       7 . The method for purifying protein according to  claim 1 , wherein the hydroxy-cholate comprises a cholate.  
   
   
       8 . The method for purifying protein according to  claim 1 , wherein the hydroxy-cholate in the eluent is maintained at a constant concentration in the elution of the target protein from the packing agent.  
   
   
       9 . The method for purifying protein according to  claim 8 , wherein the concentration of the hydroxy-cholate in the eluent is selected from a range of 0.5 through 2.5 wt %.  
   
   
       10 . The method for purifying protein according to  claim 3 , wherein the glucose dehydrogenase is produced by a microorganism belonging to the genus  Burkholderia.    
   
   
       11 . The method for purifying protein according to  claim 10 , wherein the microorganism belonging to the genus  Burkholderia  is provided by  Burkholderia cepacia  KS1 strain (FERM BP-7306).  
   
   
       12 . The method for purifying protein according to  claim 3 , wherein the glucose dehydrogenase is produced by a transformant, 
 the transformant being produced by engineering a host microorganism with a DNA from a microorganism belonging to the genus  Burkholderia  for coding the electron transfer protein and the protein active against glucose.    
   
   
       13 . The method for purifying protein according to  claim 12 , wherein the host microorganism is provided by  Pseudomonas putida.    
   
   
       14 . The method for purifying protein according to  claim 12 , wherein the host microorganism is provided by  E. coli  bacterium.  
   
   
       15 . A method for purifying glucose dehydrogenase using a combination of hydrophobic chromatography and anion exchange chromatography, wherein 
 the hydrophobic chromatography includes: a step of causing a stationary phase to hold the glucose dehydrogenase; a step of eluting unnecessary proteins; and a step of eluting the glucose dehydrogenase by using an eluent containing a hydroxy-cholate,    the anion exchange chromatography including: a step of causing a stationary phase to hold the glucose dehydrogenase; and a step of eluting the glucose dehydrogenase by using an eluent containing a hydroxy-cholate.    
   
   
       16 . The method for purifying glucose dehydrogenase according to  claim 15 , wherein concentration of the hydroxy-cholate in the eluent is varied with time in the hydrophobic chromatography, 
 concentration of the hydroxy-cholate in the eluent being kept at a constant level in the elution of the glucose dehydrogenase in the anion exchange chromatography.    
   
   
       17 . The method for purifying glucose dehydrogenase according to  claim 16 , wherein the anion exchange chromatography is carried out after the hydrophobic chromatography.  
   
   
       18 . The method for purifying glucose dehydrogenase according to  claim 15 , wherein the glucose dehydrogenase is produced by a microorganism belonging to the genus  Burkholderia.    
   
   
       19 . The method for purifying glucose dehydrogenase according to  claim 18 , wherein the microorganism belonging to the genus  Burkholderia  is provided by  Burkholderia cepacia  KS1 strain (FERM BP-7306).  
   
   
       20 . The method for purifying glucose dehydrogenase according to  claim 15 , wherein the glucose dehydrogenase is produced by a transformant, 
 the transformant being produced by engineering a host microorganism with a DNA from a microorganism belonging to the genus  Burkholderia  for coding the glucose dehydrogenase.    
   
   
       21 . The method for purifying glucose dehydrogenase according to  claim 20 , wherein the host microbe is provided by  Pseudomonas putida.    
   
   
       22 . The method for purifying glucose dehydrogenase according to  claim 20 , wherein the host microbe is provided by  E. coli  bacterium.  
   
   
       23 . The method for purifying glucose dehydrogenase according to  claim 15 , wherein the anion exchange chromatography uses an ion-exchange gel containing a quaternary ammonium group as an ion-exchange group, 
 the hydroxy-cholate being provided by a cholate.