Combinatorial enzymatic complexes

Abstract

The invention relates to the formation of novel in vivo combinatorial enzyme complexes for use in screening candidate drug agents for bioactivity.

We claim:  
     
         1 . A cell containing a composition comprising: 
 a) an exogeneous scaffold comprising at least a first binding site and a second binding site; and    b) at least a first and a second enzyme, wherein at least one of said enzymes is heterologous to said cell;    wherein said first enzyme is bound to said first binding site and said second enzyme is bound to said second binding site.    
     
     
         2 . A cell containing a composition comprising: 
 a) nucleic acid encoding an exogeneous scaffold comprising at least a first binding site and a second binding site; and    b) nucleic acid encoding at least a first and a second enzyme, wherein at least one of said enzymes is heterologous to said cell;    wherein said first enzyme is capable of being bound to said first binding site and said second enzyme is capable of being bound to said second binding site.    
     
     
         3 . A cell according to    claim 1    or    2   , wherein said scaffold comprises at least three binding sites.  
     
     
         4 . A cell according to    claim 1    or    2   , wherein said scaffold comprises at least four binding sites.  
     
     
         5 . A cell according to    claim 1    or    2   , wherein said scaffold comprises at least five binding sites.  
     
     
         6 . A cell according to    claim 1    or    2   , wherein said binding sites are on the same scaffold molecule.  
     
     
         7 . A cell according to    claim 1    or    2   , wherein said binding sites are on different scaffold molecules.  
     
     
         8 . A cell according to    claim 1    or    2   , further comprising 
 c) an exogeneous precursor.  
 
     
     
         9 . A method of screening for a bioactive agent, said method comprising: 
 a) expressing in a plurality of host cells nucleic acid encoding an exogeneous scaffold comprising at least a first binding site and a second binding site;    b) expressing in said plurality of host cells nucleic acids encoding at least a first enzyme and a second enzyme; under conditions where said nucleic acids are expressed, and said first enzyme binds to said first binding site and said second enzyme binds to said second binding site;    c) screening said host cells for a cell exhibiting an altered phenotype, wherein said altered phenotype is due to the presence of a bioactive agent.    
     
     
         10 . A method of screening for a bioactive agent, said method comprising: 
 a) expressing in a plurality of host cells a library of nucleic acids encoding a library of scaffolds, each scaffold comprising at least a first binding site and a second binding site;    b) expressing in said cells a library of nucleic acids encoding a library of enzymes;    under conditions where said nucleic acids are expressed, and at least some of said enzymes bind to said scaffolds;    c) screening said host cells for an altered phenotype.    
     
     
         11 . A method according to    claim 9   , wherein said expressing step further comprises introducing said nucleic acids into said cells.  
     
     
         12 . A method according to    claim 10   , wherein said introduction comprises retroviral infection.  
     
     
         13 . A method according to    claim 9    further comprising adding at least one exogenous precursor to said cell.  
     
     
         14 . A method according to    claim 9   , wherein said method further comprises isolating said cell exhibiting an altered phenotype.  
     
     
         15 . A method according to    claim 9    further comprising isolating said scaffold from said host cell.  
     
     
         16 . A method according to    claim 9    further comprising isolating said nucleic acid encoding said scaffold from said host cell.  
     
     
         17 . A method according to    claim 9    further comprising isolating said enzymes from said cell.  
     
     
         18 . A method according to    claim 9    further comprising isolating said nucleic acids encoding said enzymes from said cell.  
     
     
         19 . A method according to    claim 9    further comprising identifying said bioactive agent.  
     
     
         20 . A method according to    claim 9   , wherein said nucleic acids contain localization signals.  
     
     
         21 . A composition comprising: 
 a) a scaffold comprising at least a first and a second binding site; and    b) at least a first and a second enzyme; wherein said first enzyme is bound to said first binding site and said second enzyme is bound to said second binding site, wherein said enzymes do not biologically react with said scaffold or each other.    
     
     
         22 . A composition according to    claim 18    wherein said scaffold comprises a plurality of binding sites and said composition comprises a plurality of enzymes.  
     
     
         23 . A composition according to    claim 18    wherein said composition is bound to a solid support.  
     
     
         24 . A composition according to    claim 18    wherein said first binding site and said second binding sites are on the same scaffold segment.  
     
     
         25 . A composition according to    claim 18    wherein said first binding site and said second binding sites are on different segments of the scaffold.  
     
     
         26 . A method comprising: 
 a) providing a scaffold comprising at least a first binding site and a second binding site;    b) providing at least a first and a second enzyme, wherein said first enzyme is bound to said first binding site and said second enzyme is bound to said second binding site to form a enzyme complex;    c) providing said enzyme complex with at least one precursor, under conditions wherein said precursor is enzymatically altered by at least one of said enzymes to form a bioactive agent.