Purification of human troponin I

Abstract

The invention is directed to methods for purifying Troponin I, particularly recombinant Troponin I produced in a bacterial expression system. Recombinant Troponin I can be advantageously purified after reversibly protecting the free sulfhydryl groups, e.g., by forming sulfates. In a specific example, Tropnin I reacted with sodium tetrafhionate yields sulfitolyzed Tropnin I, which was purified by chromatography on an anion exchanger, followed by hydrophobic interaction chromatography. Facile deprotection of the sulfhydryl groups yields a highly purified product ready for refolding.

What is claimed is:  
     
         1 . A method of preparing Troponin I, which method comprises protecting sulfhydryl groups of Troponin I under reducing conditions.  
     
     
         2 . The method according to  claim 1 , wherein the free sulfhydryl groups are protected by sulfitolyzation.  
     
     
         3 . The method according to  claim 2 , wherein sulfitolyzation comprises reacting reduced recombinant Troponin I with sodium tetrathionate.  
     
     
         4 . The method according to  claim 1 , wherein the recombinant Troponin I is expressed in a bacterial expression system.  
     
     
         5 . The method according to  claim 4 , wherein the bacterial expression system is an  E. coli  expression system.  
     
     
         6 . The method according to  claim 1 , which further comprises purifying the sulfhydryl-protected recombinant Troponin I.  
     
     
         7 . The method according to  claim 6 , wherein the Troponin I is purified by chromatography.  
     
     
         8 . The method according to  claim 6 , which comprises purifying the Troponin I under non-reducing conditions.  
     
     
         9 . The method according to  claim 6 , which further comprises deprotecting the sulfhydryl groups.  
     
     
         10 . Troponin I comprising sulfhydryl protecting groups.  
     
     
         11 . The Troponin I of  claim 10 , which is denatured.  
     
     
         12 . The Troponin I of  claim 10 , wherein the sulfhydryl protecting groups are sulfates.  
     
     
         13 . A method of purifying Troponin I, which method comprises subjecting Troponin I comprising sulfhydryl protecting groups to chromatography.  
     
     
         14 . The method according to  claim 13 , wherein the sulfhydryl groups are protected by sulfitolyzation.  
     
     
         15 . The method according to  claim 14 , wherein sulfitolyzation comprises reacting reduced, denatured recombinant Troponin I with sodium tetrathionate.  
     
     
         16 . The method according to  claim 13 , which comprises subjecting the Troponin I to chromatography under non-reducing conditions.  
     
     
         17 . The method according to  claim 13 , wherein the Troponin I is expressed in a bacterial expression system.  
     
     
         18 . The method according to  claim 17 , wherein the bacterial expression system is an  E coli  expression system.  
     
     
         19 . The method according to  claim 13 , wherein a chromatographic support is an anion exchange column.  
     
     
         20 . The method according to  claim 19 , which further comprises chromatography on a hydrophobic interaction chromatographic support.