Methods for screening proteins using dna encoded chemical libraries as templates for enzyme catalysis

Abstract

Disclosed are methods, compositions and devices for screening a protein library for proteins having a desired activity, such as capable of catalyzing the formation of a bond between two reactants. In an exemplary embodiments, a plurality of proteins are expressed in vitro from a plurality of nucleic acids, the plurality of proteins are exposed with two single stranded oligonucleotides having complementary sequences, each oligonucleotide having a reactant and a fluorophore, the fluorescence of the protein-reactant-oligonucleotide-fluorophore complexes is detected and the complexes showing detectable fluorescence energy transfer are isolated, thereby isolating proteins having the desired enzymatic activity.

What is claimed is: 
     
         1 . A method for screening a protein library for a protein having a desired activity, the method comprising:
 a) providing, at distinct features of a solid support, a plurality of nucleic acids, each nucleic acid having a predefined sequence;   b) expressing in vitro a plurality of proteins from the plurality of nucleic acids;   c) exposing the plurality of proteins to two single-stranded oligonucleotides having complementary sequences, each single-stranded oligonucleotide having a reactant and a fluorophore;   d) detecting fluorescence of a complex having a protein having the desired activity, the reactant, the single-stranded oligonucleotide and the fluorophore; and   e) isolating the complex showing detectable fluorescence energy transfer, thereby isolating the protein having the desired activity.   
     
     
         2 . The method of  claim 1  wherein the desired activity comprises formation of a bond between two reactants. 
     
     
         3 . The method of  claim 1  wherein the protein having the desired activity specifically binds two reactants. 
     
     
         4 . The method of  claim 1  wherein the reactant is at one end of the single-stranded oligonucleotide and the fluorophore is at the other end of the single-stranded oligonucleotide. 
     
     
         5 . The method of  claim 1  wherein each distinct feature is a well of a microwell plate. 
     
     
         6 . A method for screening a protein library for a protein having a desired activity, the method comprising:
 a) providing, at distinct features of a solid support, a plurality of nucleic acids, each nucleic acid having a predefined sequence;   b) expressing in vitro a plurality of proteins from the plurality of nucleic acids;   c) exposing the plurality of proteins to a first and a second oligonucleotide each, at one end, having a first and a second reactant, respectively, wherein a protein having a desired activity binds to the first and second reactants, thereby forming a complex; and   d) ligating free ends of the first and second oligonucleotides complexed with the protein having the desired activity.   
     
     
         7 . The method of  claim 6  wherein the first and second oligonucleotides are double-stranded oligonucleotides. 
     
     
         8 . The method of  claim 6  wherein the first and second oligonucleotides are single-stranded oligonucleotides. 
     
     
         9 . The method of  claim 8  further comprising adding a helper oligonucleotide capable of bridging the single-stranded oligonucleotides bound to the protein having the desired activity. 
     
     
         10 . The method of  claim 6  wherein each distinct feature is a well of a microwell plate. 
     
     
         11 . A method for screening a protein library for a protein having a desired activity, the method comprising:
 a) culturing a host cell comprising a first nucleic acid sequence encoding a protein fused to aga-2 and a second nucleic acid sequence encoding a transcription activator-like effector fused with aga-1, so as to express the protein and the transcription activator-like effector as a first complex at the cell surface;   b) exposing the first complex to a first single-stranded oligonucleotide having a first, reactant, wherein the first single-stranded oligonucleotide binds to the transcription activator-like effector to form a second complex;   c) exposing the second complex to a second single-stranded oligonucleotide having a second reactant and a detectable label, wherein the second single-stranded oligonucleotide has, a sequence complementary to the first single-stranded oligonucleotide; and   d) detecting the detectable label at the cell surface, the presence of which indicates a protein having a desired activity.   
     
     
         12 . The method of  claim 11  wherein the host cell is a yeast cell. 
     
     
         13 . The method of  claim 11  further comprising isolating the host cell expressing the protein having the desired activity. 
     
     
         14 . The method of  claim 11  comprising culturing a plurality of host cells expressing a library of proteins. 
     
     
         15 . The method of  claim 14  wherein the library of proteins comprise a library of protein variants.