Protein having PGE2 synthase activity and use thereof

Abstract

A single protein which is the substance of the PGE2 synthesis activity in brain soluble fractions of LPS administered rats has been purified and identified. The protein has an activity of synthesizing PGE2 from PGH2, and further, has an activity of synthesizing PGE2 from arachidonic acid in combination with COX.

1 - 19 . (canceled)  
     
     
         20 . A host cell harboring a vector comprising a nucleotide sequence encoding a COX enzyme and a vector comprising a nucleotide sequence encoding a polypeptide selected from the group consisting of: 
 (a) a polypeptide comprising SEQ ID NO: 1;    (b) a polypeptide comprising SEQ ID NO: 1 with between one and thirty amino acid substitutions, deletions, additions, or insertions, wherein the polypeptide has PGE2 synthase activity;    (c) a polypeptide encoded by a DNA that hybridizes under stringent conditions to a DNA consisting of the complement of SEQ ID NO: 2; and    (d) PGES-2.    
     
     
         21 . The host cell of  claim 20 , wherein the polypeptide comprises SEQ ID NO: 1 and the COX enzyme is COX-1.  
     
     
         22 . The host cell of  claim 20 , wherein the polypeptide comprises SEQ ID NO: 1 with between one and thirty amino acid substitutions, deletions, additions, or insertions, wherein the polypeptide has PGE2 synthase activity, and the COX enzyme is COX-1.  
     
     
         23 . The host cell of  claim 20 , wherein the polypeptide is encoded by a DNA that hybridizes under stringent conditions to a DNA consisting of the complement of SEQ ID NO: 2, and the COX enzyme is COX-1.  
     
     
         24 . The host cell of  claim 20 , wherein the polypeptide is PGES-2 and the COX enzyme is COX-2.  
     
     
         25 . A method of screening for a PGH2 synthase inhibitor, the method comprising the steps of: 
 (1) contacting a test compound and PGH2 with a host cell harboring a vector comprising a nucleotide sequence encoding a polypeptide selected from the group consisting of: 
 (a) a polypeptide comprising SEQ ID NO: 1;  
 (b) a polypeptide comprising SEQ ID NO: 1 with between one and thirty amino acid substitutions, deletions, additions, or insertions, wherein the polypeptide has PGE2 synthase activity; and  
 (c) a polypeptide encoded by a DNA that hybridizes under stringent conditions to a DNA consisting of the complement of SEQ ID NO: 2;  
   (2) detecting PGE2 produced by the host cell; and    (3) selecting a test compound that reduces the level of PGE2 produced as compared with that produced in the absence of the test compound.    
     
     
         26 . The method of  claim 25 , wherein the polypeptide comprises SEQ ID NO: 1.  
     
     
         27 . The method of  claim 25 , wherein the polypeptide comprises SEQ ID NO: 1 with between one and thirty amino acid substitutions, deletions, additions, or insertions, wherein the polypeptide has PGE2 synthase activity.  
     
     
         28 . The method of  claim 25 , wherein the polypeptide is encoded by a DNA that hybridizes under stringent conditions to a DNA consisting of the complement of SEQ ID NO: 2.  
     
     
         29 . A method of screening for a PGE2 synthase inhibitor, comprising the steps of: 
 (1) contacting the host cell of  claim 21  with a test compound and arachidonic acid;    (2) detecting the level of PGE2 produced by the host cell; and    (3) selecting a test compound that reduces the level of PGE2 produced as compared with that produced in the absence of the test compound.    
     
     
         30 . A method of screening for a PGE2 synthase inhibitor, comprising the steps of: 
 (1) contacting the host cell of  claim 22  with a test compound and arachidonic acid;    (2) detecting the level of PGE2 produced by the host cell; and    (3) selecting a test compound that reduces the level of PGE2 produced as compared with that produced in the absence of the test compound.    
     
     
         31 . A method of screening for a PGE2 synthase inhibitor, comprising the steps of: 
 (1) contacting the host cell of  claim 23  with a test compound and arachidonic acid;    (2) detecting the level of PGE2 produced by the host cell; and    (3) selecting a test compound that reduces the level of PGE2 produced as compared with that produced in the absence of the test compound.    
     
     
         32 . A method of screening for a PGE2 synthase inhibitor, comprising the steps of: 
 (1) contacting the host cell of  claim 24  with a test compound and arachidonic acid:    (2) detecting the level of PGE2 produced by said host cell; and    (3) selecting a test compound that reduces the level of PGE2 produced as compared with that produced in the absence of the test compound.    
     
     
         33 . A kit for screening for a PGE2 synthase inhibitor, the kit comprising: PGH2 and a host cell harboring a vector comprising a nucleotide sequence encoding a polypeptide selected from the group consisting of: 
 (a) a polypeptide comprising SEQ ID NO: 1;    (b) a polypeptide comprising SEQ ID NO: 1 with between one and thirty amino acid substitutions, deletions, additions, or insertions, wherein the polypeptide has PGE2 synthase activity; and    (c) a polypeptide encoded by a DNA that hybridizes under stringent conditions to a DNA consisting of the complement of SEQ ID NO: 2.    
     
     
         34 . The kit of  claim 33 , wherein the polypeptide comprises SEQ ID NO: 1.  
     
     
         35 . The kit of  claim 33 , wherein the polypeptide comprises SEQ ID NO: 1 with between one and thirty amino acid substitutions, deletions, additions, or insertions, wherein the polypeptide has PGE2 synthase activity.  
     
     
         36 . The kit of  claim 33 , wherein the polypeptide is encoded by a DNA that hybridizes under stringent conditions to a DNA consisting of the complement of SEQ ID NO: 2.  
     
     
         37 . A kit for screening for a PGE2 synthase inhibitor, the kit comprising the host cell of  claim 21  and arachidonic acid.  
     
     
         38 . A kit for screening for a PGE2 synthase inhibitor, the kit comprising the host cell of  claim 22  and arachidonic acid.  
     
     
         39 . A kit for screening for a PGE2 synthase inhibitor, the kit comprising the host cell of  claim 23  and arachidonic acid.  
     
     
         40 . A kit for screening for a PGE2 synthase inhibitor, the kit comprising the host cell of  claim 24  and arachidonic acid.