Generation of specific binding partners binding to (poly)peptides encoded by genomic DNA fragments or ESTs

Abstract

The present invention relates to the generation of specific binding partners binding to (poly)peptides encoded by genomic DNA fragments or ESTs. The (poly)peptides are expressed as part of fusion proteins which are forming inclusion bodies on expression in host cells. The inclusion bodies are used to generate binding partners which bind specifically to said (poly)peptides. The specific binding partners, in particular immunoglobulins or fragments thereof, are useful for analysis and functional characterisation of proteins encoded by nucleic acid sequences comprising the corresponding genomic DNA fragments or ESTs. The invention further relates to nucleic acid molecules, vectors and host cells to be used in the methods of the present invention. The invention further relates to the use of fusion proteins comprising the first N-terminal domain of the geneIII protein of filamentous phage as fusion partner for the expression of a (poly)peptide/protein fused to said fusion partner, and to methods for the expression of (poly)peptide/proteins.

1 . A method for generating a specific binding partner to a (poly)peptide which is encoded by a nucleic acid sequence comprised in a genomic DNA fragment or an expressed sequence tag (EST) comprising: 
 a) expressing a nucleic acid molecule encoding a fusion protein in a host cell under conditions that allow the formation of inclusion bodies comprising said fusion protein, wherein said fusion protein comprises 
 aa) a (poly)peptide/protein fusion partner which is deposited in inclusion bodies when expressed in said host cell under said conditions, and  
 ab) said (poly)peptide;  
   b) isolating said inclusion bodies; and    c) generating a specific binding partner that binds specifically to said (poly)peptide.    
     
     
         2 . The method of  claim 1 , wherein said fusion protein comprises said fusion partner as N-terminal portion and said (poly)peptide as C-terminal portion.  
     
     
         3 . The method of claims  1  or  2 , wherein said fusion protein further comprises a (poly)peptide linker linking said fusion partner and said (poly)peptide.  
     
     
         4 . The method of  claim 3 , wherein said linker comprises a cleavage signal.  
     
     
         5 . The method of any one of  claims 1  to  4 , wherein said genomic DNA fragment or said EST is obtained from a prokaryotic organism or from a virus.  
     
     
         6 . The method of  claim 5 , wherein said prokaryotic organism or virus is a pathogen.  
     
     
         7 . The method of any one of  claims 1  to  4 , wherein said genomic DNA fragment or said EST is obtained from a eukaryotic organism.  
     
     
         8 . The method of  claim 7 , wherein said genomic DNA fragment or EST is obtained from a non-mammalian species.  
     
     
         9 . The method of  claim 7 , wherein said genomic DNA fragment or EST is obtained from a mammalian species.  
     
     
         10 . The method of  claim 9 , wherein said mammalian species is human.  
     
     
         11 . The method of any one of  claims 1  to  10 , wherein said nucleic acid is expressed under conditions allowing over-expression of said fusion protein.  
     
     
         12 . The method of any one of  claims 1  to  11 , wherein said host cell is a eukaryotic cell.  
     
     
         13 . The method of  claim 12 , wherein said eukaryotic cell is a yeast or insect cell.  
     
     
         14 . The method of any one of  claims 1  to  11 , wherein said host cell is a prokaryotic cell.  
     
     
         15 . The method of  claim 14 , wherein said prokaryotic cell is a bacterial cell.  
     
     
         16 . The method of  claim 15 , wherein said bacterial cell is an  E. coli  cell.  
     
     
         17 . The method of claims  15  or  16 , wherein said fusion protein is expressed in the cytosol.  
     
     
         18 . The method of  claim 17 , wherein said fusion partner contains at least one disulfide bond.  
     
     
         19 . The method of claims  17  or  18 , wherein said fusion partner is a secreted protein, and wherein said nucleic acid does not comprise a nucleic acid sequence encoding a signal sequence for the transport of the fusion protein to the periplasm.  
     
     
         20 . The method of any one of  claims 1  to  19 , wherein said fusion partner is an endogenous (poly)peptide/protein of said host cell.  
     
     
         21 . The method of any one of  claims 1  to  19 , wherein said fusion partner is a (poly)peptide/protein foreign to said host cell.  
     
     
         22 . The method of any one of  claims 1  to  21 , wherein said fusion partner is taken from the list of  E. coli  maltose-binding protein,  E. coli  RNAse II,  E. coli  alkaline phosphatase,  E. coli  phosholipase A,  E. coli  β-lactamase,  E. coli  thioredoxin, human procathepsin B, porcine interferon, and T5 DNA polymerase.  
     
     
         23 . The method of  claim 21 , wherein said host cell is  E. coli  and said fusion partner comprises the first N-terminal domain of the geneIII protein of a filamentous phage.  
     
     
         24 . The method of  claim 23 , wherein said fusion partner consists of amino acids 1 to 82 of the geneIII protein.  
     
     
         25 . The method of any one of  claims 1  to  24 , wherein step b) further comprises the step of (i) solubilising said fusion protein under suitable conditions.  
     
     
         26 . The method of  claim 25 , wherein step b) further comprises the step of (ii) refolding said fusion protein under suitable conditions.  
     
     
         27 . The method of claims  25  or  26 , wherein said fusion protein further comprises a (poly)peptide linker linking said fusion partner and said (poly)peptide, wherein said linker comprises a cleavage signal, and wherein step b) further comprises the steps of (iii) cleaving said fusion protein between said fusion partner and said (poly)peptide, and 
 (iv) isolating said (poly)peptide in free form.  
 
     
     
         28 . The method of any one of  claims 25  to  27 , further comprising the step of purifying said fusion protein or said (poly)peptide in free form.  
     
     
         29 . The method of any one of  claims 1  to  28 , wherein said specific binding partner is an immunoglobulin or a fragment thereof.  
     
     
         30 . The method of  claim 29 , wherein said immunoglobulin is generated by (i) immunisation of an animal with said inclusion bodies, said fusion protein or said (poly)peptide, and (ii) by selecting an immunoglobulin produced by said animal which specifically binds to said inclusion bodies, said fusion protein or said (poly)peptide.  
     
     
         31 . The method of  claim 29 , wherein said immunoglobulin or fragment thereof is generated by selecting a member of a recombinant library of immunoglobulins or fragments thereof which specifically binds to said inclusion bodies, said fusion protein or said (poly)peptide.  
     
     
         32 . The method of  claim 31 , wherein said library is displayed on the surface of a replicable genetic package.  
     
     
         33 . The method of  claim 32 , wherein said replicable genetic package is a filamentous phage.  
     
     
         34 . A nucleic acid molecule encoding a fusion protein comprising aa) the first N-terminal domain of the geneIII protein of filamentous phage and ab) a (poly)peptide which is encoded by a nucleic acid sequence comprised in a genomic DNA fragment or an expressed sequence tag (EST), wherein said nucleic acid molecule does not comprise a nucleic acid sequence encoding a signal sequence for the transport of the fusion protein to the periplasm of a bacterial host cell.  
     
     
         35 . A vector comprising a nucleic acid molecule of  claim 34 .  
     
     
         36 . The vector of  claim 35  which is an expression vector.  
     
     
         37 . A host cell comprising a nucleic acid of  claim 34  or a vector of claims  35  or  36 .  
     
     
         38 . The host cell of  claim 37  which is an  E. coli  cell.  
     
     
         39 . The use of a fusion protein comprising the first N-terminal domain of the geneIII protein of filamentous phage as fusion partner for the expression of a (poly)peptide/protein fused to said fusion partner, wherein said fusion protein is obtained in the form of inclusion bodies.  
     
     
         40 . A method for the expression of a (poly)peptide/protein comprising: 
 a) expressing a nucleic acid molecule encoding a fusion protein in a host cell under conditions that allow the formation of inclusion bodies comprising said fusion protein, wherein said fusion protein comprises 
 aa) the first N-terminal domain of the geneIII protein of filamentous phage, and  
 ab) said (poly)peptide/protein.  
   
     
     
         41 . The method of  claim 40  further comprising the steps of 
 b) isolating said inclusion bodies; and  
 c) solubilising said fusion protein under suitable conditions.