Cellular Libraries of Peptide Sequences (CLiPS) and Methods of Using the Same

Abstract

The present invention provides compositions including peptide display scaffolds that present at least one candidate peptide and at least one detectable moiety in at least one of the N-terminal and C-terminal candidate peptide presenting domains that when expressed in a cell are accessible at a surface of the cell outermembrane. In addition, the present invention also provides kits and methods for screening a library of cells presenting the candidate peptides in peptide display scaffolds to identify a ligand for an enzyme.

1 - 46 . (canceled) 
     
     
         47 . A method of identifying a substrate for a target protease, the method comprising:
 contacting a plurality of cells with a background mixture,   wherein the background mixture does not include the target protease and wherein each of the plurality of cells expresses a peptide display scaffold; and   enriching the plurality of cells for cells that are not cleaved by background mixture,   wherein the peptide display scaffold comprises a fusion protein having the structure [D 1 -C 1 ]-TM-[D 2 ],   wherein TM is a circularly permuted transmembrane protein, wherein C 1  comprises a candidate substrate of the target protease, wherein D 1  is a first detectable moiety and is a heterologous peptide or polypeptide, wherein D 2  is a second detectable moiety and is a heterologous peptide or polypeptide, wherein D 1  and D 2  are not the same; and wherein D 1 , D 2 , and C 1  are exposed at an extracellular surface of the cell outer membrane.   
     
     
         48 . The method of  claim 47 , wherein the step of enriching the plurality of cells comprises:
 depleting the plurality of cells that are cleaved by the background mixture.   
     
     
         49 . The method of  claim 47  comprising:
 contacting the enriched plurality of cells with the target protease. 
 
     
     
         50 . The method of  claim 49  comprising:
 enriching the enriched plurality of cells for cells that are cleaved by the target protease. 
 
     
     
         51 . The method of  claim 50 , wherein the step of enriching the enriched plurality of cells comprises:
 isolating the cells from the enriched plurality of cells that are cleaved by the target protease; and   amplifying the isolated cells.   
     
     
         52 . The method of  claim 47  comprising:
 detecting the presence or absence of a D 1  signal and a D2 signal for at least one cell, wherein said detecting indicates whether C 1  is cleaved by the background mixture. 
 
     
     
         53 . The method of  claim 49  comprising:
 detecting the presence or absence of a D 1  signal and a D2 signal for at least one cell, wherein said detecting indicates whether C 1  is cleaved by the target protease. 
 
     
     
         54 . The method of  claim 47 , wherein the background mixture comprises a cellular lysate or a tissue extract. 
     
     
         55 . The method of  claim 47 , wherein the fusion protein comprises at least one linker, wherein the linker is between C 1  and TM, or wherein the linker is between D 2  and TM. 
     
     
         56 . The method of  claim 47 , wherein at least one of detectable moieties D 1  and D 2  comprises an affinity tag. 
     
     
         57 . The method of  claim 47 , wherein at least one of detectable moieties D 1  and D 2  comprises a fluorescent protein. 
     
     
         58 . The method of  claim 52 , wherein the detecting step comprises detecting the presence of a detectable signal of D 1  and detecting the absence of a detectable signal of D 2  that is above a background level of a detectable D 2  signal. 
     
     
         59 . The method of  claim 53 , wherein the detecting step comprises detecting the presence of a detectable signal of D 1  and detecting the absence of a detectable signal of D 2  that is above a background level of a detectable D 2  signal. 
     
     
         60 . The method of  claim 47 , wherein the D 1  and C 1  are located at an N-terminal domain of the fusion protein and the D 2  is located at a C-terminal domain of the fusion protein. 
     
     
         61 . The method of  claim 47 , wherein the D 1  and C 1  are located at a C-terminal domain of the fusion protein and the D 2  is located at an N-terminal domain of the fusion protein. 
     
     
         62 . The method of  claim 47 , wherein the circularly permuted transmembrane protein is a circularly permuted bacterial outer membrane selected from the group consisting of: a circularly permuted OmpX, a circularly permuted OmpA, and a circularly permuted OmpT. 
     
     
         63 . A method of identifying a substrate for a target protease, the method comprising:
 contacting a plurality of cells with a background mixture, wherein the background mixture does not include the target protease and wherein each of the plurality of cells expresses a peptide display scaffold;   enriching the plurality of cells that are not cleaved by the background mixture;   contacting the enriched plurality of cells with the target protease;   isolating the cells from the enriched plurality of cells that are cleaved by the target protease; and   amplifying the isolated cells,   wherein the peptide display scaffold comprises a fusion protein having the structure [D 1 -C 1 ] TM-[D 2 ],   wherein TM is a circularly permuted transmembrane protein, wherein C 1  comprises a candidate substrate for the target protease, wherein D 1  is a first detectable moiety and is a heterologous peptide or polypeptide, wherein D 2  is a second detectable moiety and is a heterologous peptide or polypeptide, wherein D 1  and D 2  are not the same; and wherein D 1 , D 2 , and C 1  are exposed at an extracellular surface of the cell outer membrane.   
     
     
         64 . The method of  claim 63 , wherein the step of enriching the plurality of cells that are not cleaved by the target protease comprises:
 depleting the plurality of cells of cells that are cleaved by the background mixture.   
     
     
         65 . The method of  claim 63  comprising;
 detecting the presence or absence of a D 1  signal and a D 2  signal for at least one cell, wherein said detecting indicates whether C 1  is cleaved by the background mixture. 
 
     
     
         66 . The method of  claim 63  comprising;
 detecting the presence or absence of a D 1  signal and a D 2  signal for at least one cell, wherein said detecting indicates whether C 1  is cleaved by the target protease 
 
     
     
         67 . The method of  claim 63 , wherein the background mixture comprises a cellular lysate or a tissue extract. 
     
     
         68 . The method of  claim 63 , wherein the circularly permuted transmembrane protein is a circularly permuted bacterial outer membrane selected from the group consisting of: a circularly permuted OmpX, a circularly permuted OmpA, and a circularly permuted OmpT.