Tryparedoxin, expression plasmid, process of production, method of use, test kit, and pharmaceutical composition

Abstract

The present invention provides novel enzymes, tryparedoxins, their isolation from Crithidia fasciculata, a method for the production thereof in genetically transformed bacteria, and their use as molecular targets for the discovery of trypanocidal drugs

We claim  
     
         1 . A protein capable of transferring reductive equivalents of trypanothione (T(SH) 2 ) to a peroxiredoxin.  
     
     
         2 . A protein according to  claim 1  prepared or isolated from a species of the family Trypanosomatidae  
     
     
         3 . A protein according to  claim 1  comprising the following amino acid sequence SEQ ID NO 1 or a part thereof:  
       
         
           
                 
                 
                 
               
                     
                 
                     
                     G   K   V   L   F   F   Y   F     S   A    
                   15 
                 
                   1 
                   GGS AAR CTR GTS TTC TTC TAC TTC TCC GCG 
                 
                     
                                         S   W   C   P   P    
                 
                     
                                       AGC TGG TGC CCG CCG 
                 
                     
                 
                     
                      C   R   G   F   T   P   O   L   I   E   
                   30 
                 
                   46 
                   TGC GCG GGC TTC ACG CCG CAG CTG ATC GAG 
                 
                     
                                          F   Y   D   K   F   
                 
                     
                                       TTC TAC GAC AAG TTC 
                 
                     
                 
                     
                     H   E   S   K    N   F   E   V   V   F  
                   45 
                 
                   91 
                   CAC GAG TCG AAG AAC TTC GAG GTT GTG TTC 
                 
                     
                                        C   S   W   D   E  
                 
                     
                                       TGC ACG TGG GAC GAG 
                 
                     
                 
                     
                    E   E   D   G   F   R   G   Y   F   A  
                   60 
                 
                   136 
                   GAG GAG GAC GGC TTT GCG GGC TAC TTC GCG 
                 
                     
                                        K    M   P   W   L    
                 
                     
                                       AAG ATG CCG TGG CTT 
                 
                     
                 
                     
                     A   V   P   F   A   O   S   E   A   V    
                   75 
                 
                   181 
                   GCG GTG CCG TTT GCG CAG AGC GAG GCG GTG 
                 
                     
                                         O   K    L   S    K    
                 
                     
                                       CAG AAG CTG TCG AAS 
                 
                     
                 
                     
                     H   F   N   V   E    80 
                 
                   226 
                   CAS TTC AAC GTC GAG 
                 
                     
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         4 . A protein having the amino acid sequence of SEQ ID NO 1 according to  claim 3 .  
     
     
         5 . A protein according to  claim 1  having a molecular weight of 15 kDa to 19 kDa  
     
     
         6 . A protein according to  claim 1  characterized by a WCPPC motif (FIG. 3) and capable of catalyzing the reduction of protein disulfides by trypanothione (T(SH) 2 ).  
     
     
         7 . A protein according to  claim 6  wherein said protein disulfide is a peroxiredoxin.  
     
     
         8 . A protein according to  claim 7  wherein said peroxiredoxin is tryparedoxin peroxidase (TXNP x ).  
     
     
         9 . A protein having the amino acid sequence of SEQ ID NO 2 (FIG. 3).  
     
     
         10 . A protein capable of transferring reductive equivalents of T(SH) 2  to a peroxiredoxin 
 (a) having the amino acid sequence SEQ ID NO 2 (FIG. 3, positions 1 to 150).                                  M   S   G   L    K   K   F   F    P  Y    14     1   ATG TCA GGG CTG AAG AAG TTC TTC CCT TAC                                  S    T   N   V                                 AGC ACA AAC GTG                L   K   G   D   A   A   D   I   A   L    28     43   CTG AAG GGT GCT GCT GCG GAT ATC GCG CTC                                  P   S   L   A                                  CCC TCG CTG GCG                G   K   T   V   F   F   Y   F   S   A    42     85   GGC AAG ACC GTA TTC TTC TAC TTC TCC GCG                                  S    W   C   P                                   AGC TGG TGC CCG                  P  C    R   A   F   T   P   Q   L   I    56     127   CCG TGC CGG GCC TTC ACG CCG  CAG CTG  ATC                                  E   F   Y   K                                  GAT TTT TAC AAG                A   H   A   E   K   K   N   F   E   V    70     169   GCC CAC GCG GAG AAG AAG AAC TTC GAG GTG                                  M   L   I   F                                  ATG CTC ATC TCC                W   D   E   S   A   E   D   F   K   K    84     211   TGG GAT GAG TCA GCA GAG GAC TTT AAG GAC                                  Y   Y  A   K                                   TAC TAC GCG AAG                M   P   W   L   A   L   P   F   E   D    98     253   ATG CCG TGG CTG GCA TTG CCG TTT GAA GAC                                  R   K   G   M                                  CGC AAA GGG ATG                E   F   L   T   T   G   F   D   V   K    112     295   GAG TTC TTG ACG ACC GGC TTC GAT GTG AAG                                  S   I   P   T                                  TCG ATC CCA ACC                L   V   G   V   E   A   D   S   G   N    126     337   TTG GTG GGC GTC GAG GCG GAC TCG GGA AAC                                  I   I   T   T                                  ATC ATC ACA ACG                Q   A   R   T   M   V   V   K   D   P    140     378   CAG GCG CGT ACG ATG GTG GTG AAG GAC CCG                                  E   A   K   D                                  GAA GCA AAG GAT                F   P   W   P   N   V   E   A   K   K    150     421   TTT CCG TGG CCC AAC GTG GAG GCC AAG AAG                                                                                             TAA                                                                              or    (b) having an amino acid sequence which is homologous to said sequence of (a), has about the same number of amino acids, and is encoded by an oligonucleotide, the complementary strand of which is hybridizable (at 1 M NaCl concentration and a temperature of at least 25° C.) with an oligonucleotide which encodes a protein having the amino acid sequenced SEQ ID NO 1 or SEQ ID NO 2.    
     
     
         11 . A protein according to  claim 10  (b) having an amino acid sequence which is homologous to said sequence of  claim 10  (a) by at least 70%.  
     
     
         12 . The protein according to  claim 11  having an amino acid sequence which is homologous to said sequence by at least 75%.  
     
     
         13 . A protein according to  claim 10  having an amino acid sequence which differs in its number of amino acids for SEQ ID NO 2 by up to 30 amino acids.  
     
     
         14 . A protein according to  claim 10  having an amino acid sequence which differs in its number of amino acids for SEQ ID NO 2 by up to 25 amino acids.  
     
     
         15 . A protein according to  claim 10  having an amino acid sequence which differs in its number of amino acids for SEQ ID NO 2 by up to 20 amino acids.  
     
     
         16 . A plasmid for the expression of a protein according to  claim 1  and comprising a nucleic acid encoding said protein.  
     
     
         17 . A plasmid containing a DNA sequence encoding a protein according to  claim 10  (tryparedoxin=TXN).  
     
     
         18 . A plasmid according to  claim 17  containing a DNA sequence encoding a protein of  Crithidia fasciculata.    
     
     
         19 . A plasmid for the expression of an isolatable, functionally active derivative of a protein according to  claim 10  (tryparedoxin=TXN), wherein the plasmid contains a DNA sequence which encodes said derivative  
     
     
         20 . A plasmid containing a DNA sequence encoding a functionally active derivative of a protein according to  claim 10  (tryparedoxin=TXN) wherein the tryparedoxin is derivatised by a His tag.  
     
     
         21 . A process for producing a protein of  claim 10  comprising the steps of culturing a host transformed with DNA encoding said protein and isolating said protein from said host or its culture medium.  
     
     
         22 . The process of  claim 21  wherein said DNA encoding said protein comprises SEQ ID NO 1 or a part thereof.  
     
     
         23 . The process of  claim 21  wherein said DNA encoding said protein comprises SEQ ID NO 2 or a part thereof.  
     
     
         24 . The process of  claim 21  wherein said host is transformed with a plasmid containing said DNA sequence.  
     
     
         25 . A process according to  claim 21  wherein the host is selected from the group consisting of bacteria, fungi, yeast, plant cells, insect cells, mammalian cells, and cell cultures.  
     
     
         26 . A process according to  claim 21  wherein the host is  Escherichia coli.    
     
     
         27 . A method for identifying inhibitors of the activity of the protein of  claim 1  comprising the steps of measuring the activity of said protein in the presence and  
     
     
         27 . A method for identifying inhibitors of the activity of the protein of  claim 1  comprising the steps of measuring the activity of said protein in the presence and absence of said inhibitor and detecting a decrease in the activity of said protein in the presence of said inhibitor.  
     
     
         28 . A test kit for testing the catalytic activity of a protein according to  claim 1  comprising trypanothione (T(SH) 2 ), trypanothione reductase (TR), a tryparedoxin peroxidase (TXNPx), the protein the activity of which is to be tested, and a hydroperoxide as indicator enzyme, mediator, and substrate, respectively  
     
     
         29 . A pharmaceutical composition having trypanocidal activity and comprising an inhibitory substance inhibiting the catalytic activity of a protein according to  claim 1.