Recombinant expression of S-layer proteins

Abstract

The invention concerns a process for the recombinant production of S-layer proteins in gram-negative host cells. Furthermore the nucleotide sequence of a new S-layer gene and processes for the production of modified S-layer proteins are disclosed.

1 . A recombinant S-layer protein, wherein said protein is coded by an isolated nucleic acid selected from the group consisting of 
 (i) a nucleic acid comprising a nucleotide sequence from position 1 to 3684 of SEQ ID NO:1,    (ii) a nucleic acid comprising a nucleotide sequence which encodes an amino acid sequence according to SEQ ID NO:2, and    (iii) a nucleic acid comprising a nucleotide sequence which hybridizes with at least one of the nucleic acids of (i) or (ii) under stringent conditions,    wherein the nucleic acid contains at least one peptide or polypeptide-coding insertion within the region coding for the S-layer protein.    
     
     
         2 . The protein according to  claim 1 , wherein the insertion site is located at position 582, 878, 917, 2504 and/or 2649 of the nucleotide sequence shown in SEQ ID NO:1.  
     
     
         3 . A recombinant S-layer structure, wherein said structure contains at least one protein according to  claim 1  as a subunit.  
     
     
         4 . The S-layer structure as claimed in  claim 3 , further comprising at least one unmodified S-layer protein as a subunit.  
     
     
         5 . The S-layer structure as claimed in  claim 3 , wherein it comprises several layers which are linked covalently or by affinity binding.  
     
     
         6 . The recombinant S-layer protein as claimed in  claim 1 , wherein the insertions code for streptavidin.  
     
     
         7 . The recombinant S-layer protein as claimed in  claim 1 , wherein the insertions code for immunogenic epitopes from herpes viruses, in particular herpes virus 6 or FMDV.  
     
     
         8 . The recombinant S-layer protein as claimed in  claim 1 , wherein the insertions code for enzymes such as polyhydroxybutyric acid synthase or bacterial luciferase.  
     
     
         9 . The recombinant S-layer protein as claimed in  claim 1 , wherein the insertions code for cytokines such as interleukins, interferons or tumour necrosis factors.  
     
     
         10 . The recombinant S-layer protein as claimed in  claim 1 , wherein the insertions code for antibody-binding proteins such as protein A or protein G.  
     
     
         11 . The recombinant S-layer protein as claimed in  claim 1 , wherein the insertions code for antigenic epitopes which bind cytokines or endotoxins.  
     
     
         12 . The recombinant S-layer protein as claimed in  claim 1 , wherein the insertions code for metal-binding epitopes.  
     
     
         13 . The recombinant S-layer protein as claimed in  claim 1 , wherein the insertions are selected from nucleotide sequences which code for cysteine residues, regions with several charged amino acids or Tyr residues, DNA-binding epitopes, metal binding epitopes, immunogenic epitopes, allergenic epitopes, antigenic epitopes, streptavidin, enzymes, cytokines or antibody-binding proteins.  
     
     
         14 . An S-layer protein, wherein said protein is coded by a nucleic acid selected from the group consisting of: 
 (i) a nucleic acid comprising a nucleotide sequence from position 1 to 2763 of SEQ ID NO:5,    (ii) a nucleic acid comprising a nucleotide sequence corresponding to the nucleic acid from (i) within the scope of the degeneracy of the genetic code, and    (iii) a nucleic acid comprising a nucleotide sequence which hybridizes with at least one of the nucleic acids of (i) or (ii) under stringent conditions,    wherein the nucleic acid contains at least one peptide or polypeptide-coding insertion within the region encoding the S-layer protein.    
     
     
         15 . A recombinant S-layer structure, wherein said structure contains at least one recombinant S-layer protein according to  claim 14  as a subunit.  
     
     
         16 . The recombinant S-layer protein as claimed in  claim 14 , wherein the insertions are selected from nucleotide sequences which code for cysteine residues, regions with several charged amino acids or Tyr residues, DNA-binding epitopes, metal-binding epitopes, immunogenic epitopes, allergenic epitopes, antigenic epitopes, streptavidin, enzymes, cytokines or antibody-binding proteins.  
     
     
         17 . The recombinant S-layer protein as claimed in  claim 14 , wherein the insertions code for streptavidin.  
     
     
         18 . The recombinant S-layer protein as claimed in  claim 14 , wherein the insertions code for immunogenic epitopes from herpes viruses, in particular herpes virus 6 or FMDV.  
     
     
         19 . The recombinant S-layer protein as claimed in  claim 14 , wherein the insertions code for enzymes such as polyhydroxybutyric acid synthase or bacterial luciferase.  
     
     
         20 . The recombinant S-layer protein as claimed in  claim 14 , wherein the insertions code for cytokines such as interleukins, interferons or tumour necrosis factors.  
     
     
         21 . The recombinant S-layer protein as claimed in  claim 14 , wherein the insertions code for antibody-binding proteins such as protein A or protein G.  
     
     
         22 . The recombinant S-layer protein as claimed in  claim 14 , wherein the insertions code for antigenic epitopes which bind cytokines or endotoxins.  
     
     
         23 . The recombinant S-layer protein as claimed in  claim 14 , wherein the insertions code for metal-binding epitopes.