Polypeptide display libraries and methods of making and using thereof

Abstract

Disclosed herein are expression vectors which display a passenger polypeptide on the outer surface of a biological entity. As disclosed herein the displayed passenger polypeptide is capable of interacting or binding with a given ligand. Also disclosed are methods of making and using the expression vectors. N/C terminal fusion expression vectors and methods of making and using are also disclosed.

1 - 20 . (canceled) 
     
     
         21 . A method of identifying a candidate substrate for a target protease or peptidase, the method comprising:
 contacting a cell with the target protease or peptidase,
 wherein the cell comprises a passenger polypeptide and a carrier protein, 
 wherein the carrier protein is a circularly permuted variant of a native outer membrane protein, wherein a native N-terminus and a native C-terminus of the native outer membrane protein are joined by a linker, wherein the carrier protein comprises a non-native N-terminus and a non-native C-terminus derived from an extracellular loop of the native outer membrane protein, 
 wherein the passenger polypeptide is linked or fused to the non-native N-terminus or to the non-native C-terminus of the carrier protein, 
 wherein the passenger polypeptide comprises the candidate substrate of the target protease or peptidase, a first label fused to a free terminus of the passenger polypeptide, 
   wherein the passenger polypeptide and the candidate substrate are exposed at an extracellular surface of a outer membrane of the cell; and   
       detecting the presence or absence of a signal from the first label. 
     
     
         22 . The method of  claim 21 , wherein the step of detecting the presence or absence of the signal from the first label indicates an interaction between the target protease or peptidase and the candidate substrate. 
     
     
         23 . The method of  claim 22 , wherein the interaction between the target protease or peptidase and the candidate substrate comprises cleavage of the candidate substrate by the target protease or peptidase to produce the presence or absence of the signal from the first label. 
     
     
         24 . The method of  claim 21 , wherein the step of detecting the presence or absence of the signal from the first label comprises:
 detecting the signal by flow cytometry, fluorescence, or microscopy.   
     
     
         25 . The method of  claim 21 , wherein the first label comprises an affinity tag. 
     
     
         26 . The method of  claim 21 , wherein the first label comprises a fluorescent label. 
     
     
         27 . The method of  claim 21 , wherein the cell is a bacterial cell, a yeast cell, or a mammalian cell. 
     
     
         28 . The method of  claim 27 , wherein the bacterial cell is an  Escherichia coli  cell. 
     
     
         29 . The method of  claim 21 , wherein the native outer membrane protein is a bacterial outer membrane protein selected from the group consisting of: OmpA or OmpX. 
     
     
         30 . The method of  claim 29 , wherein the non-native N-terminus and the non-native C-terminus are derived from a first extracellular loop of OmpA. 
     
     
         31 . The method of  claim 29 , wherein the non-native N-terminus and the non-native C-terminus are derived from a second extracellular loop of OmpX. 
     
     
         32 . The method of  claim 29 , wherein the non-native N-terminus and the non-native C-terminus are derived from a third extracellular loop of OmpX. 
     
     
         33 . The method of  claim 21 , wherein the non-native N-terminus is positioned extracellularly. 
     
     
         34 . The method of  claim 21 , wherein the non-native C-terminus is positioned extracellularly. 
     
     
         35 . The method of  claim 21 , wherein the first label is C-terminal to the passenger polypeptide. 
     
     
         36 . The method of  claim 21 , wherein the first label is N-terminal to the passenger polypeptide. 
     
     
         37 . The method of  claim 21 , wherein the cell comprises a second label. 
     
     
         38 . The method of  claim 37 , wherein the second label is a fluorescent label. 
     
     
         39 . The method of  claim 37  comprising:
 detecting the presence or absence of a signal from the second label. 
 
     
     
         40 . The method of  claim 39 , wherein the step of detecting the presence or absence of the signal from the second label comprises:
 detecting the signal by flow cytometry, fluorescence, or microscopy.