Methods for Purifying Pertussis Toxin and Peptides Useful Therefor

Abstract

The present invention relates to reagents and methods for purifying pertussis toxin (PT).

1 - 51 . (canceled) 
     
     
         52 . A method for isolating pertussis toxin comprising contacting pertussis toxin with an immobilized peptide comprising an amino acid sequence selected from the group consisting of: 
       
         
           
                 
                 
               
                     
                   (SEQ ID NO.: 67) 
                 
                     
                   RSSHCRHRNCHTITRGNMRIETPNNIRKDAK; 
                 
                     
                     
                 
                     
                   (SEQ ID NO.: 68) 
                 
                     
                   RSSHCRHRNCHTITRGNMRIETPNNIRKDA; 
                 
                     
                     
                 
                     
                   (SEQ ID NO.: 69) 
                 
                     
                   RSTMNTNRMDIQRLMTNHVKRDSSPGSIDA; 
                 
                     
                     
                 
                     
                   (SEQ ID NO.: 70) 
                 
                     
                   RSNVIPLNEVWYDTGWDRPHRSRLSIDDDA; 
                 
                     
                     
                 
                     
                   (SEQ ID NO.: 71) 
                 
                     
                   RSWRDTRKLHMRHYFPLAIDSYWDHTLRDA; 
                 
                     
                     
                 
                     
                   (SEQ ID NO.: 72) 
                 
                     
                   SGCVKKDELCARWDLVCCEPLECIYTSELYATCGK; 
                 
                     
                     
                 
                     
                   (SEQ ID NO.: 73) 
                 
                     
                   SGCVKKDELCARWDLVCCEPLECIYTSELYATCG; 
                 
                     
                     
                 
                     
                   (SEQ ID NO.: 74) 
                 
                     
                   SGCVKKDELCELAVDECCEPLECFQMGHGFKRCG; 
                 
                     
                     
                 
                     
                   (SEQ ID NO.: 75) 
                 
                     
                   SGCVKKDELCSQSVPMCCEPLECKWFNENYGICGS; 
                 
                     
                     
                 
                     
                   (SEQ ID NO.: 76) 
                 
                     
                   SGCVKKDELCELAIDECCEPLECTKGDLGFRKCG; 
                 
                     
                   and, 
                 
                     
                     
                 
                     
                   (SEQ ID NO 14) 
                 
                     
                   CVKKDELCXXXXXXCCEPLECXXXXXXXXXC, 
                 
                     
                   where X is any amino acid. 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         53 . The method of  claim 52  wherein the immobilized peptide comprises the amino acid sequence RSNVIPLNEVWYDTGWDRPHRSRLSIDDDA (SEQ ID NO.: 70). 
     
     
         54 . The method of  claim 52  wherein the pertussis toxin is isolated from a fermentation supernatant. 
     
     
         55 . The method of  claim 52  wherein the peptide is immobilized by binding to a solid support. 
     
     
         56 . The method of  claim 55  wherein the solid support is selected from selected from the group consisting of a column, chromatographic media, column material, bead, test tube, microtiter dish, solid particle, sepharose, agarose, microchip, silicon, silicon-glass, gold, and a membrane. 
     
     
         57 . The method of  claim 52  wherein the pertussis toxin is eluted from the peptide using a buffer comprising glycine, carbonate or magnesium. 
     
     
         58 . The method of  claim 57  wherein the buffer comprises glycine at about pH 2.5. 
     
     
         59 . The method of  claim 57  wherein the buffer comprises carbonate at about pH 10.5. 
     
     
         60 . The method of  claim 57  wherein the buffer comprises MgCl 2 . 
     
     
         61 . The method of  claim 56  wherein the solid support is a sepharose bead comprising 300-400 pmol of the peptide. 
     
     
         62 . The method of  claim 52  comprising a complex of a peptide and a solid support. 
     
     
         63 . The method of  claim 62  wherein the pertussis toxin is immobilized to a complex of a peptide and a solid support, the method further comprising eluting the pertussis toxin from the complex and regenerating the complex. 
     
     
         64 . The method of  claim 63  wherein the complex is regenerated using HCl. 
     
     
         65 . The method of  claim 52  wherein the immobilized peptide comprises the amino acid sequence RSNVIPLNEVWYDTGWDRPHRSRLSIDDDA (SEQ ID NO.: 70) and the pertussis toxin is eluted from the peptide using a buffer comprising glycine at about pH 2.5 or carbonate at about pH 10.5.