Method

Abstract

The present invention relates to novel methods of producing proteins in which one or more domains are full length and correctly folded and which are each tagged at either the N— or C-terminus with one or more marker moieties and arrays containing such proteins, as well as the use of such proteins in arrays for rapid screening.

1 . A method for producing one or more proteins in which one or more domains are full length and correctly folded and which are each tagged at either the N— or C-terminus with one or more marker moieties, said method comprising: 
 (a) providing one or more DNA molecules having an open reading frame encoding said proteins together with 5′ and/or 3′ untranslated regions;    (b) treating said DNA molecules first with a 5′ to 3′- or 3′ to 5′-nuclease to generate a set of nested deletions followed by converting the nested set of deletions into blunt-ended double stranded DNA molecules;    (c) cloning the fragments generated in (b) into an expression vector containing a coding sequence for one or more 5′ or 3′ marker moieties; and    (d) expressing said encoded proteins.    
     
     
         2 . The method as claimed in  claim 1 , wherein at (b) said blunt-ended double stranded DNA molecules are generated by treating said nested deletions with a single-strand nuclease under conditions that allow removal of said 5′ or 3′ untranslated regions including the start or stop codons of said open reading frame.  
     
     
         3 . The method as claimed in  claim 1 , wherein said DNA molecules of (a) are provided as double stranded cDNA synthesis products.  
     
     
         4 . The method as claimed in  claim 3  wherein after (a) and prior to (b) said DNA molecules are specifically protected at the 5′ or 3′ end from nuclease digestion.  
     
     
         5 . The method as claimed in  claim 3 , wherein said DNA molecules of (a) are biotinylated.  
     
     
         6 . The method as claimed in  claim 1 , wherein said DNA molecules of (a) are provided as linearised plasmids.  
     
     
         7 . The method as claimed in  claim 6  wherein said plasmids are derived from a cDNA library.  
     
     
         8 . The method as claimed in  claim 6 , wherein plasmids are obtained by propagating bacterial cells containing said library through growth of individual cultures each containing a single clonal member of the library.  
     
     
         9 . A tagged protein prepared by a method as defined in  claim 1 .  
     
     
         10 . A library of tagged proteins produced by the method of  claim 1 .  
     
     
         11 . An array of proteins in which one or more domains are full length and correctly folded and which are tagged at the C-terminus with one or more marker moieties, said marker moieties being appended to the encoding DNA molecules in a sequence independent manner.  
     
     
         12 . An array comprising proteins prepared by a method as defined in  claim 1 .  
     
     
         13 . An array as claimed in  claim 12 , wherein the components of the array are immobilized.  
     
     
         14 . An array as claimed in  claim 13 , wherein the components of the array are immobilized by means of a tag moiety.  
     
     
         15 . An array as claimed in  claim 13 , wherein said immobilization is to a solid surface.  
     
     
         16 . An array as claimed in  claim 11 , wherein the components of the array are immobilized.  
     
     
         17 . An array as claimed in  claim 16 , wherein the components of the array are immobilized by means of a tag moiety.  
     
     
         18 . An array as claimed in  claim 16 , wherein said immobilization is to a solid surface.