Novel method for purification of recombinant proteins

Abstract

Purification of poly-amino acid-tagged recombinant proteins has been improved by the use of a carboxymethylated aspartate ligand complexed with a third-block transition metal having an oxidation state of 2 + and a coordination number of 6. A method for synthesizing the metal ion-CM-Asp complex is also described. Further, the metal ion-CM-Asp complex can be used for screening protein function.

1 . An immobilized metal ion affinity chromatography purification method for purification of a recombinant proteins, said method comprising: 
 (a) providing carboxymethylated aspartate ligand complexed with a transition metal ion in a 2 +  oxidation state, having a coordination number of 6;    (b) loading a mixture of cell lysate comprising a recombinant protein having a polyhistidine tail to bind with said ligand; and    (c) eluting said recombinant protein with a suitable elutant to obtain a purified recombinant protein.    
   
   
       2 . The method, according to  claim 1 , wherein said transition metal-complexed carboxymethylated aspartate ligand forms a carboxymethylated aspartate chelating matrix which comprises said transition metal and a polymer matrix.  
   
   
       3 . The method, according to  claim 2 , wherein said transition metal is connected to said polymer matrix by a linking arm and a functional linking group.  
   
   
       4 . The method, according to  claim 3 , wherein said linking arm is selected from the group consisting of —CH 2 CH(OH)CH 2 —, —CH 2 (OH)CH 2 —O—CH 2 CH(OH)CH 2 —, —(CH 2 ) 4 NHCH 2 CH(OH)CH 2 —, and —{CH 2 ) 2 NHCH 2 CH(OH)CH 2 —.  
   
   
       5 . The method, according to  claim 3 , wherein said functional linking group is selected from the group consisting of O, S, and NH.  
   
   
       6 . The method, according to  claim 2 , wherein said polymer matrix is agarose.  
   
   
       7 . The method, according to  claim 2 , wherein said carboxymethylated aspartate chelating matrix has the structure  
     
       
         
         
             
             
         
       
     
     wherein: 
 R 4 —R 5 —R 6 ═H  
 M=transition metal ion in a 2 +  oxidation state with a coordination number of 6;  
 R 1 =a linking arm connecting the nitrogen atom of CM-Asp with R 2 ;  
 R 2 =a functional linking group through which CM-Asp linking arm R 1  is connected to R 3 ; and  
 R 3 =a polymer matrix  
 
   
   
       8 . The method, according to  claim 2 , wherein said carboxymethylated aspartate chelating matrix has the structure  
     
       
         
         
             
             
         
       
     
     wherein: 
 R 1 —R 2 —R 3 ═H;  
 M=transition metal ion in a 2 +  oxidation state with a coordination number of 6;  
 R 4 =a linking arm connecting the methylene carbon atom of the carboxymethyl group of CM-Asp with R 5 ;  
 R 5 =a functional linking group through which CM-Asp linking arm R 4  is connected to R 6 ;  
 and R 6 =a polymer matrix.  
 
   
   
       9 . An immobilized metal ion affinity chromatography complex comprising a carboxymethylated aspartate ligand and a transition metal complexed thereto, wherein said transition metal ion has a 2 +  oxidation state and a coordination number of 6.  
   
   
       10 . The complex, according to  claim 9 , wherein said complex has the structure:  
     
       
         
         
             
             
         
       
     
     wherein: 
 R 4 —R 5 —R 6 ═H  
 M=transition metal ion in a 2 +  oxidation state with a coordination number of 6;  
 R 1 =a linking arm connecting the nitrogen atom of CM-Asp with R 2 ;  
 R 2 =a functional linking group through which CM-Asp ticking arm R 1 , is connected to R 3 ; and  
 R 3 =a polymer matrix  
 
   
   
       11 . The method, according to  claim 10 , wherein said polymer matrix comprises a polymer matrix suitable for use in affinity or gel chromatography.  
   
   
       12 . The complex, according to  claim 10 , wherein 
 M=Fe 2+ , Co 2+ , Ni 2+ , Cu 2+ , or Zn 2+ ;    R 1 ═—CH 2 CH(OH)CH 2 —,—CH 2 (OH)CH 2 —O—CH 2 CH(OH)CH 2 — or —(CH 2 )NHCH 2 CH(OH)CH 2 —.    R 2 ═O, S, or NH; and    R 3 =agarose or polystyrene.    
   
   
       13 . The complex, according to  claim 12 , wherein 
 M=Co 2+ ;    R 1 ═CH 2 CH(OH)CH 2 ;    R 2 ═O; and    R 3 =agarose, cross-linked or polystyrene    
   
   
       14 - 21 . (canceled)  
   
   
       22 . A method for synthesizing carboxymethylated aspartate chelating matrices, said method comprising the steps: 
 (a) Michael addition of the a-amino function of monoprotected α, ω-diamino acids to maleic acid;    (b) deprotecting the co-amino functionality; and    (c) attaching the chelator primary amine molecule to a solid matrix.    
   
   
       23 . A method for screening for protein function on a microtiter plate or filter, said method comprising the steps: 
 (a) immobilizing a complex of  claim 1  to the plate or filter;    (b) binding said immobilized complex to the protein for which the function is being screened; and    (c) performing an assay for protein function on the bound protein.