Novel methods for obtaining, identifying and applying nucleic acid sequences and (poly)peptides which increase the expression yields of periplasmic proteins in functional form

Abstract

The present invention relates to a method for obtaining nucleic acid sequences encoding (poly)peptides which increase the expression yields of periplasmic proteins in functional form upon co-expression of said (poly)peptides and said periplasmic proteins. The invention also provides a method for the identification of said (poly)peptides. Furthermore, the present invention relates to a method for increasing the expression yields of periplasmic proteins in functional form by co-expressing (poly)peptides, for example Skp, FkpA, or a homolog of Skp or FkpA, in bacteria.

1 . A method for obtaining a nucleic acid sequence comprising a (poly)peptide coding sequence, which increases the expression yield of a periplasmic protein in functional form in bacteria upon co-expression of said periplasmic protein and said (poly)peptide, comprising the steps of: 
 (a) providing a collection of host cells wherein each cell contains 
 (i) a first nucleic acid sequence out of a collection of nucleic acid sequences, and  
 (ii) a second nucleic acid sequence encoding said periplasmic protein;  
   (b) causing or allowing expression of 
 (i) (poly)peptides expressible from said collection of nucleic acid sequences, and  
 (ii) said periplasmic protein expressible from said second nucleic acid sequence;  
   (c) screening or selecting for a host cell expressing said periplasmic protein with increased functional yield;    (d) optionally, repeating step (c) one or more times;    (e) obtaining said first nucleic acid sequence contained in said host cell.    
     
     
         2 . The method of  claim 1 , further comprising the step of identifying a (poly)peptide coding sequence comprised in said first nucleic acid sequence.  
     
     
         3 . The method of claims  1  or  2 , wherein said first nucleic acid sequence is or is derived from genomic DNA or mRNA of an organism, or cDNA.  
     
     
         4 . The method of anyone of  claims 1  to  3 , wherein said first nucleic acid sequence comprises an at least partially randomized sequence.  
     
     
         5 . The method of anyone of  claims 1  to  4 , wherein 
 (a) said first nucleic acid sequence is comprised in a vector which can be packaged in a filamentous phage particle, and  
 (b) said periplasmic protein is a fusion protein of at least part of a filamentous phage coat protein and a further protein;  
 and wherein in the course of said expression a collection of filamentous phage particles displaying said further protein is produced from said collection of host cells.  
 
     
     
         6 . The method of anyone of  claims 1  to  5  wherein said further protein comprises at least a domain of the immunoglobulin superfamily, and preferably of the immunoglobulin family.  
     
     
         7 . The method of  claim 6  wherein said further protein is an immunoglobulin fragment taken from the list of Fv, scFv, disulphide-linked Fv, and Fab fragments.  
     
     
         8 . A method for identifying a (poly)peptide which increases the expression yield of a periplasmic protein in functional form in bacteria upon co-expression of said periplasmic protein and said (poly)peptide, comprising the steps of: 
 (a) identifying a nucleic acid sequence or a (poly)peptide coding sequence according to a method of anyone of  claims 1  to  7 , and    (b) deducing a (poly)peptide therefrom.    
     
     
         9 . A method for increasing the expression of a periplasmic protein in functional form in a bacterial host cell, characterized by co-expressing said periplasmic protein and a (poly)peptide identified by the method according to  claim 8 .  
     
     
         10 . The method of  claim 9 , wherein said periplasmic protein is a member of a collection of periplasmic proteins expressed in a collection of host cells.  
     
     
         11 . The method of claims  9  or  10  wherein said (poly)peptide is the  E. coli  protein Skp or a homolog thereof.  
     
     
         12 . The method of claims  9  or  10  wherein said (poly)peptide is the  E. coli  protein FkpA or a homolog thereof.  
     
     
         13 . The method of anyone of  claims 9  to  12  wherein said periplasmic protein ia a fusion protein of at least part of a filamentous phage coat protein and a further protein.  
     
     
         14 . The method of anyone of  claims 9  to  13  wherein said further protein comprises at least a domain of the immunoglobulin superfamily, and preferably of the immunoglobulin family.  
     
     
         15 . The method of  claim 14 , wherein the further protein is an immunoglobulin fragment taken from the list of Fv, scFv, disulphide-linked Fv, and Fab fragment.