Fusion proteins, DNA molecules, vectors, and host cells useful for measuring protease activity

Abstract

The present invention relates to novel fusion proteins, DNA molecules encoding the same, vectors comprising the DNA molecules, and host cells containing the vectors for use in measuring protease activity using a novel transcriptional assay. This invention also relates to a method for determining the inhibitory activity of a compound against a protease and to a method for comparing the activity of two proteases which recognize the same cleavage site. Kits for assaying protease activity comprising DNA molecules encoding the fusion protein substrates of this invention are also contemplated.

We claim:  
     
         1 . A fusion protein comprising: 
 a. a protease cleavage site;    b. a ligand binding domain; and    c. a DNA binding domain;    wherein the association of a ligand with said fusion protein mediates the binding of the fusion protein to a ligand-responsive element (“LRE”) operatively linked to a reporter gene; and    wherein the fusion protein also comprises an expression modulator domain or associates with a second protein having an expression modulator domain, wherein said expression modulator domain regulates the transcription of said reporter gene.    
     
     
         2 . The fusion protein according to  claim 1 , wherein said protease cleavage site is recognized by HCV NS3 protease.  
     
     
         3 . The fusion protein according to  claim 1 , wherein said protease cleavage site is recognized by HIV aspartyl protease.  
     
     
         4 . The fusion protein according to  claim 1 , wherein said DNA binding domain is selected from a DNA binding domain of a steroid/thyroid superfamily receptor.  
     
     
         5 . The fusion protein according to  claim 1 , wherein said DNA binding domain is from the ecdysone receptor and wherein said ecdysone receptor requires association with a protein binding partner to enable binding to DNA.  
     
     
         6 . The fusion protein according to  claim 5 , wherein said DNA binding domain is modified such that the P-box region has the amino acid sequence Seq. ID. No. 24.  
     
     
         7 . The fusion protein according to  claim 1 , wherein said expression modulator domain is the activation domain of the VP16 protein.  
     
     
         8 . The fusion protein according to  claim 1  having the sequence of SEQ ID NO: 18.  
     
     
         9 . The fusion protein according to  claim 1 , wherein the amino acid sequence of the protease cleavage site is selected from the group consisting of Seq. ID. Nos. 1-10.  
     
     
         10 . A DNA molecule encoding a fusion protein according to any one of  claims 1  to  9 .  
     
     
         11 . The DNA molecule according to  claim 6 , wherein said DNA sequence is SEQ ID No. 17.  
     
     
         12 . A vector comprising the DNA molecule according to  claim 10  or  11 .  
     
     
         13 . A host cell transformed with a vector according to  claim 12 .  
     
     
         14 . A method of producing a fusion protein according to any one of  claims 1  to  9 , comprising the steps of culturing a host cell according to  claim 13  under conditions which cause expression of said fusion protein.  
     
     
         15 . The host cell according to  claim 13  further comprising a DNA molecule comprising: 
 a. an LRE which binds to said DNA binding domain of said fusion protein, wherein said binding is modulated by the presence of a ligand and is optionally modulated by the presence of a protein binding partner of said fusion protein;  
 b. a promoter which is modulated by said expression modulating domain of said fusion protein; and  
 c. a reporter gene, the expression of which is controlled by said promoter.  
 
     
     
         16 . The host cell according to  claim 15 , wherein said promoter is a mammalian heat shock promoter.  
     
     
         17 . The host cell according to  claim 15 , wherein said reporter gene is the luciferase gene.  
     
     
         18 . The host cell according to  claim 15 , additionally comprising a DNA molecule which encodes a protein binding partner necessary for activating said DNA binding domain of said fusion protein.  
     
     
         19 . A method for assaying protease activity in vitro comprising the steps of: 
 a. incubating a fusion protein according to any one of  claims 1  to  5  in an in vitro transcription extract with a protease capable of cleaving said fusion protein at said protease cleavage site and a DNA molecule, wherein said DNA molecules comprise (i) a LRE which binds to the DNA binding domain of said fusion protein; (ii) a promoter which is modulated by the expression modulating domain of the fusion protein; and (iii) a reporter gene, the expression of which is controlled by the promoter, wherein said protease cleavage site of said fusion protein can be cleaved by said protease whose activity is being assayed;    b. adding to said incubation a ligand which associates with said fusion protein, wherein said ligand is required for said fusion protein to (i) bind to said LRE or to bind to a protein binding partner; and (ii) modulate the transcription of said reporter gene; and    c. quantifying the gene product produced from said reporter gene.    
     
     
         20 . A method for assaying protease activity in a cell comprising the steps of: 
 a. culturing a host cell according to any one of  claims 15  to  18  under conditions which cause expression of said fusion protein and, if present, said protein binding partner, wherein said host cell expresses the protease to be assayed and wherein said protease cleavage site of said fusion protein can be cleaved by said protease;    b. adding to said host cell culture a ligand which associates with and regulates the activity of said fusion protein, wherein said ligand is required for said fusion protein to (i) bind to said LRE operatively linked to a reporter gene or to bind to a protein binding partner; and (ii) modulate the transcription of said reporter gene;    c. quantifying the gene product produced from said reporter gene.    
     
     
         21 . A method for determining the inhibitory activity of a compound against a protease comprising the steps of: 
 a. culturing in a first culture a host cell according to any one of  claims 15  to  18  in the absence of said compound under conditions which cause expression of said fusion protein, wherein said host cell expresses the protease to be assayed and wherein said protease cleavage site of said fusion protein can be cleaved by said protease;    b. culturing in a second culture the host cell used in step a. in the presence of said compound under conditions which cause expression of said fusion protein;    c. adding to said first and to said second host cell cultures a ligand, wherein said ligand is required for said fusion protein to (i) bind to said LRE operatively linked to a reporter gene or to bind to a protein binding partner; and (ii) modulate the transcription of said reporter gene which associates of said fusion protein;    d. comparing the amount of gene product produced from said reporter gene in said first host cell culture and said second host cell culture.    
     
     
         22 . The method according to  claim 21 , comprising the additional steps of: 
 a. culturing in a third culture a host cell according to any one of  claims 15  to  18  under conditions which cause expression of said fusion protein and wherein said host cell does not express a protease which recognizes the protease cleavage site in said fusion protein; and, as part of step d., comparing the amount of gene product produced from said reporter gene in said host cell culture with the amount of gene product produced from said reporter gene in said first and second host cell cultures.    
     
     
         23 . A method of comparing the activity of two proteases which recognize the same cleavage site, said method comprising the steps of: 
 a. culturing a first host cell according to any one of  claims 15  to  18  under conditions which cause expression of said fusion protein and, if present, said protein binding partner, wherein said first host cell expresses a first protease which is capable of cleaving said protease cleavage site of said fusion protein;    b. culturing a second host cell according to any one of  claims 15  to  18  under conditions which cause expression of said fusion protein and, if present, said protein binding partner, wherein said second host cell expresses a second protease which is capable of cleaving said protease cleavage site of said fusion protein;    c. adding to said first and to said second host cell culture a ligand, wherein said ligand is required for said fusion protein to (i) bind to said LRE operatively linked to a reporter gene or to bind to a protein binding partner; and (ii) modulate the transcription of said reporter gene; and    d. comparing the amount of gene product produced from said reporter gene in said first host cell culture and said second host cell culture.    
     
     
         24 . The method according to  claim 23 , wherein said first and said second proteases are forms of HIV protease.  
     
     
         25 . A kit for assaying protease activity comprising: 
 a. a DNA molecule according to  claim 10  or  11  or a vector according to  claim 12;     b. a DNA molecule comprising: 
 i. a LRE which is capable of being bound to said DNA binding domain of said fusion protein encoded by the DNA molecule of a.;  
 ii. a promoter which is capable of being modulated by said expression modulating domain of said fusion protein encoded by the DNA molecule of a.; and  
 iii. a reporter gene, the expression of which is controlled by said promoter;  
   c. a ligand, wherein said ligand is required for said fusion protein to (i) bind to said LRE operatively linked to a reporter gene or to bind to a protein binding partner; and (ii) modulate the transcription of said reporter gene; and    d. instructions for using said kit to assay protease activity.    
     
     
         26 . The kit according to  claim 25 , wherein said DNA molecule of a. and said DNA molecule of b. are both present in a host cell.  
     
     
         27 . The kit according to  claim 26  further including a DNA molecule or a vector comprising a DNA molecule encoding a protein binding partner of said fusion protein.