Methods for Enhancing Bacterial Cell Display of Proteins and Peptides

Abstract

Methods of making and using bacterial display polypeptide libraries using circularly permuted OmpX (CPX) variants are disclosed. The invention further relates to methods for enhancing the display of proteins and peptides at the surface of bacteria by optimizing linkers and incorporating mutations at positions 165 and 166 of CPX.

1 .- 42 . (canceled) 
     
     
         43 . A method of screening a library of polypeptides for the ability to bind to a target molecule, the method comprising:
 a) providing a polypeptide display library comprising a circularly permuted OmpX (CPX) variant carrying a plurality of passenger polypeptides displayed on bacterial cells;   b) contacting the plurality of passenger polypeptides with a target molecule, and   c) identifying at least one displayed passenger polypeptide that binds to the target molecule,   wherein the CPX variant comprises:
 a linker joining the native N-terminus and native C-terminus of OmpX, 
 wherein the CPX variant comprises a non-native N-terminus and a non-native C-terminus, 
 wherein the linker is 4-8 residues in length and comprises a sequence X-Z n , 
 wherein X is an amino acid selected from the group consisting of: serine, threonine, and proline, 
 wherein each Z is independently any amino acid, and n is 2 to 4, and 
 wherein at least one of the Z residues is independently selected from the group consisting of: lysine, arginine, glutamine, asparagine, or histidine. 
   
     
     
         44 . The method of  claim 43 , wherein the target molecule is selected from the group consisting of a receptor, a ligand, an antibody, an antigen, an enzyme, a transporter, a substrate, an inhibitor, an activator, a cofactor, a drug, a nucleic acid, a lipid, a carbohydrate, a glycoprotein, a small organic molecule, and an inorganic molecule. 
     
     
         45 . The method of  claim 43 , wherein said target molecule comprises a detectable label, wherein identifying the target molecule bound to at least one passenger polypeptide comprises detecting the label attached to said target molecule. 
     
     
         46 . The method of  claim 43 , wherein the first residue of the linker of the CPX variant is a glycine. 
     
     
         47 . The method of  claim 43 , wherein the linker of the CPX variant comprises at least two basic residues. 
     
     
         48 . The method of  claim 43 , wherein the linker of the CPX variant comprises two arginine residues. 
     
     
         49 . The method of  claim 43 , wherein the linker of the CPX variant comprises two lysine residues. 
     
     
         50 . The method of  claim 43 , wherein the linker of the CPX variant comprises at least one arginine residue and at least one lysine residue. 
     
     
         51 . The method of  claim 43 , wherein the linker of the CPX variant is 5 residues in length. 
     
     
         52 . The method of  claim 43 , wherein the linker of the CPX variant is 6 residues in length. 
     
     
         53 . The method of  claim 52 , wherein the first residue of the linker of the CPX variant is a glycine and the third and sixth residues of the linker of the CPX variant are selected from the group consisting of arginine, lysine, serine, histidine, glutamine, and asparagine. 
     
     
         54 . The method of  claim 43 , wherein the CPX variant comprises one or more mutations that increase the display efficiency of a passenger peptide compared to the CPX variant in the absence of the mutations, wherein at least one mutation is at a position corresponding to A165 or G166 of the native OmpX protein consisting of SEQ ID NO: 1. 
     
     
         55 . The method of  claim 54 , wherein the CPX variant comprises one or more mutations selected from the group consisting of an A165V mutation, an A165L mutation, an A1651 mutation, an A165F mutation, a G166S mutation, a G166A mutation and combinations thereof. 
     
     
         56 . The method of  claim 43 , wherein the CPX variant comprises a passenger polypeptide fused to the non-native N-terminus of the CPX variant. 
     
     
         57 . The method of  claim 56 , wherein the CPX variant comprises a linker between the non-native N-terminus of the CPX variant and the passenger polypeptide. 
     
     
         58 . The method of  claim 43 , wherein the CPX variant comprises a passenger polypeptide fused to the non-native C-terminus of the CPX variant. 
     
     
         59 . The method of  claim 58 , wherein the CPX variant comprises a linker between the non-native C-terminus of the CPX variant and the passenger polypeptide. 
     
     
         60 . The method of  claim 43 , wherein the CPX variant comprises a first passenger polypeptide fused to the non-native N-terminus of the CPX variant and a second passenger polypeptide fused to the non-native C-terminus of the CPX variant. 
     
     
         61 . The method of  claim 60 , wherein the first passenger polypeptide or the second passenger polypeptide comprises a detectable label. 
     
     
         62 . The method of  claim 61 , wherein both the first passenger polypeptide and the second passenger polypeptide comprise detectable labels. 
     
     
         63 . The method of  claim 62 , wherein the first passenger polypeptide comprises a different detectable label than the second passenger polypeptide. 
     
     
         64 . The method of  claim 43 , wherein the linker of the CPX variant comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs: 6, 8-10, 12-14, 17, 18, 20, 21, and 24-26.