Glutathionylspermidine synthetase and processes for recovery and use thereof

Abstract

The present invention describes an enzyme showing glutathionylspermidine synthetase-activity and being distinct from known enzymes with similar activities in several physicochemical parameters, a novel process to isolate said enzyme from Crithidia fasciculata, tools for the production thereof in genetically transformed organisms, and its use as a molecular target for the discovery of trypanocidal drugs.

We claim:  
     
         1 . A protein catalyzing the synthesis of glutathionylspermidine having a pH optimum of said synthesis of about pH 7.5 and having a molecular weight of 78,000±3,000 Da.  
     
     
         2 . A protein according to  claim 1  comprising an amino acid sequence selected from the group consisting of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, and SEQ ID NO 7 as shown in FIG. 1.  
     
     
         3 . A protein according to  claim 1  isolated from a species of the family of trypanosomatids (Trypanosomatidae).  
     
     
         4 . A protein according to  claim 3  wherein said species is selected from the group consisting of Trypanosoma sp., Leishmania sp., Herpetomonas sp., Leptomonas sp., Blastocrithidia sp., Crithidia sp., and Phytomotnas sp.  
     
     
         5 . A protein according to  claim 4  wherein said species is  C. fasciculata.    
     
     
         6 . A protein according to  claim 1  produced by the method comprising the steps of: 
 (a) culturing a host cell transformed with DNA selected from DNA encoding any one of the amino acid sequences SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, and SEQ ID NO 7 as shown in FIG. 1 and DNA encoding the nucleotide sequence SEQ ID No 8 shown in FIG. 2, and,  
 (b) isolating said protein from said host cell or its growth medium.  
 
     
     
         7 . A protein according to  claim 1  comprising a partial deduced amino acid sequence selected from the group consisting of SEQ ID NO 8 as shown in FIG. 2, and sequences homologous to said SEQ ID NO 8 having the same number of amino acids as SEQ ID NO 8 and being identical to SEQ ID NO 8 in more than 70% of the amino acid residues.  
     
     
         8 . A protein according to  claim 7  wherein said sequence is identical to SEQ ID NO 8 in more than 75% of the amino acid residues.  
     
     
         9 . A protein according to  claim 1  encoded in part by a partial DNA sequence selected from the group consisting of SEQ ID NO 9 shown in FIG. 2 and other DNA sequences having the same number of nucleotides and being identical to SEQ ID NO 9 in more than 70% of the nucleotides.  
     
     
         10 . A protein according to  claim 9  wherein said other DNA sequence is identical to SEQ ID NO 9 in more than 75% of the nucleotides.  
     
     
         11 . A protein according to  claim 9  wherein the complementary strand of said other DNA sequence hybridizes to SEQ ID NO 9 at a temperature of at least 25° C. and at a NaCl concentration of 1M.  
     
     
         12 . A process for recovering a protein according to  claim 1  comprising the steps of 
 (a) homogenizing cells belonging to Trypanosomatidae;  
 (b) extracting the resulting homogenate with an aqueous multi-phase system;  
 (c) separating the resulting phase containing the protein; and  
 (d) optionally, isolating the protein from said phase of (c).  
 
     
     
         13 . A process according to  claim 12  wherein the extraction step (b) is carried out with an aqueous two-phase system.  
     
     
         14 . A process according to  claim 12  wherein one of the aqueous phases of the multi-phase system contains at least one organic polymer, and another phase of the multi-phase system is an aqueous solution of at least one salt.  
     
     
         15 . A process according to  claim 14  wherein said organic polymer comprises polyethylene glycol (PEG) having a molecular weight of more than 1500 Da.  
     
     
         16 . A process according to  claim 15  wherein said PEG has a molecular weight more than 6000 Da.  
     
     
         17 . A process according to  claim 14  wherein said one aqueous phase contains PEG having a molecular weight of about 6000 Da, and the other aqueous phase is a phosphate solution.  
     
     
         18 . A process according to  claim 17  wherein said phosphate solution has a pH of about 7.  
     
     
         19 . A process according to  claim 7  comprising the step, before step (d) of extracting the protein contained in said phase separated in step (c) by means of another aqueous phase optionally followed by at least one additional step of extracting the protein from the phase with an aqueous phase up to substantially complete separation of enzyme activities cleaving ATP, other than the protein wanted.  
     
     
         20 . A process according to  claim 19  wherein the other aqueous phase is free of polyethylene glycol (PEG) or has a lower PEG concentration than any phase of the multi-phase system of step (b).  
     
     
         21 . A process according to  claim 19  comprising the step of extracting the phase separated in step (c) by lowering the pH thereof.  
     
     
         22 . A process according to  claim 21  wherein said pH is lowered to pH 6 or below.  
     
     
         23 . A process according to  claim 19  wherein said another aqueous phase contains at least one organic polymer or at least one dissolved salt.  
     
     
         24 . A process according to  claim 7  wherein said cells are selected from the group consisting of Trypanosoma, Leishmania, Herpetomonas, Leptomonas, Blastocrithidia, Crithidia, and Phytomotnas.  
     
     
         25 . A process according to  claim 24  wherein said cells are  Crithidia fasciculata  cells.  
     
     
         26 . A process according to  claim 25  wherein said cells are a-pathogenic.  
     
     
         27 . A method of identifying compounds having trypanocidal activity (GspS inhibitors) comprising the steps of 
 (a) contacting the protein of  claim 7  with said compound in an aqueous phase and    (b) measuring inhibition of the activity of the protein.    
     
     
         28 . The method according to  claim 27  wherein the activity of the protein is measured by measuring ATP hydrolysis.  
     
     
         29 . GspS inhibitors identified by the method of  claim 27 .  
     
     
         30 . Isolated GspS inhibitors of  claim 29 .  
     
     
         31 . Pharmaceutical composition comprising a GspS inhibitor according to  claim 29  and at least one of a carrier and an adjuvant.