Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1998-12-02
2004-08-17
Minnifield, N. M. (Department: 1645)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S320100, C435S069300, C435S069500, C435S069700, C435S070100, C435S071100, C435S252300, C435S071200, C536S023100, C536S023500, C536S023600
Reexamination Certificate
active
06777202
ABSTRACT:
The present invention concerns processes for the recombinant production of S-layer proteins and modified S-layer proteins in gram-negative host cells.
Crystalline bacterial cell surface layers (S-layers) form the outermost cell wall component in many eubacteria and most of the archaebacteria (Sleytr et al. (1988), Crystalline Bacterial Cell Surface Layers, “Springer Verlag Berlin”; Messner and Sleytr, Adv. Microb. Physiol. 33 (1992), 213-275). Most of the presently known S-layer proteins are composed of identical proteins or glycoproteins which have apparent molecular weights in the range of 40,000 to 220,000. The components of S-layers are self-assembling and most of the lattices have an oblique (p2), quadratic (p4) or hexagonal (p6) symmetry. The functions of bacterial S-layers are still not completely understood but due to their location on the cell surface the porous crystalline S-layers probably serve mainly as protective coatings, molecular sieves or to promote cell adhesion and surface recognition.
Genetic data and sequence information are known for various S-layer genes from microorganisms. A review may be found in Peyret et al., Mol. Microbiol. 9 (1993), 97-109. Explicit reference is made to these data. The sequence of the sbsA gene coding for the S-layer protein of B.stearothermophilus PV72 and a process for cloning it are stated in Kuen et al. (Gene 145 (1994), 115-120). B.stearothermophilus PV72 is a gram-positive bacterium which is covered with a hexagonally arranged S-layer. The main component of the S-layer is a 128 kd protein which is the most frequent protein in the cell with a proportion of about 15% relative to the total protein components. Various strains of B.stearothermophilus have been characterized which differ with regard to the type of the S-layer lattice, the molecular weight and glycosilation of the S-layer components (Messner and Sleytr (1992), supra).
The German Patent Application P 44 25 527.6 discloses the signal peptide-coding section of the S-layer gene from B.stearothermophilus and the amino acid sequence derived therefrom. The cleavage site between the signal peptide and the mature protein is located between position 30 and 31 of the amino acid sequence. The signal peptide-coding nucleic acid can be operatively linked to a protein-coding nucleic acid and can be used for the recombinant production of proteins in a process in which a transformed host cell is provided, the host cell is cultured under conditions which lead to an expression of the nucleic acid and to production and secretion of the polypeptide coded thereby and the resulting polypeptide is isolated from the culture medium. Prokaryotic organisms are preferably used as host cells in particular gram-positive organisms of the genus bacillus.
Surprisingly it was found that the recombinant production of S-layer proteins is not only possible in gram-positive prokaryotic host cells but also in gram-negative prokaryotic host cells. In this case the S-layer protein is not formed in the interior of the host cell in the form of ordered inclusion bodies but rather unexpectedly in the form of ordered monomolecular layers.
Hence one subject matter of the present invention is a process for the recombinant production of S-layer proteins characterized in that (a) a gram-negative prokaryotic host cell is provided which is transformed with a nucleic acid coding for an S-layer protein selected from (i) a nucleic acid which comprises the nucleotide sequence shown in SEQ ID NO. 1 from position 1 to 3684 optionally without the section coding for the signal peptide, (ii) a nucleic acid which comprises 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 which comprises a nucleotide sequence which hybridizes with the nucleic acids from (i) or/and (ii) under stringent conditions; (b) the host cell is cultured under conditions which lead to an expression of the nucleic acid and to production of the polypeptide coded thereby and (c) the resulting polypeptide is isolated from the host cell.
The term “stringent hybridization” is understood within the sense of the present invention to mean that a hybridization still also occurs after washing at 55° C., preferably 60° C. in an aqueous low salt buffer (e.g. 0.2× SSC) (see also Sambrook et al. (1989), Molecular Cloning. A Laboratory Manual).
The process according to the invention is carried out in gram-negative prokaryotic host cells. In this process an ordered S-layer protein structure is surprisingly obtained in the cell interior. Enterobacteria, in particular
E. coli
, are preferably used as host cells.
The
E. coli
strain pop2135 which was deposited on 31.01.1996 at the “Deutsche Sammlung von Mikroorganismen und Zelikulturen GmbH”, Mascheroder Weg 1b, D 38124 Braunschweig under the file number DSM 10509 is particularly preferred
The process according to the invention can also be used to isolate recombinant S-layer proteins. For this one uses a nucleic acid coding for the S-layer protein which contains one or several insertions which code for peptide or polypeptide sequences. These insertions can, on the one hand, only code for peptides with a few amino acids e.g. 1-25 amino acids. On the other hand, the insertions can also code for larger polypeptides of for example up to 1000 amino acids and preferably up to 500 amino acids without loss of the ability of the S-layer protein to form a correctly folded structure. In addition to the insertions the recombinant S-layer protein can also have amino acid substitutions, in particular substitutions of individual amino acids in the region of the insertion sites as well as optionally deletions of individual amino acids or short amino acid sections of up to 30 amino acids.
Regions between the positions 1-1200 and 2200-3000 of the nucleotide sequence shown in SEQ ID NO.1 are preferred as insertion sites for polypeptide-coding sequences. Particularly preferred insertion sites are the NruI cleavage site at position 582, the PvuII cleavage site at position 878, the SnaB-I cleavage site at position 917, the PvuII cleavage site at position 2504 and the PvuII cleavage site at position 2649. It was already possible to demonstrate the insertion of a nucleic acid coding for streptavidin into the NruI cleavage site at position 581.
The peptide or polypeptide-coding insertions are preferably selected from nucleotide sequences which code for cysteine residues, regions with several charged amino acids, e.g. Arg, Lys, Asp or Glu, or Tyr residues, DNA-binding epitopes, antigenic, allergenic or immunogenic epitopes, metal-binding epitopes, streptavidin, enzymes, cytokines or antibody-binding proteins.
A particularly preferred example of an insertion into the nucleic acid coding for the S-layer protein is a nucleotide sequence coding for streptavidin. In this manner it is possible to obtain universal carrier molecules which are suitable for coupling biotinylated reagents and for detection in immunological or hybridization test procedures.
A further preferred example of insertions are antigenic, allergenic or immunogenic epitopes e.g. epitopes from pathogenic microorganisms such as bacteria, fungi, parasites etc. and viruses, or epitopes from plants or epitopes against endogenous substances e.g. cytokines as well as against toxins in particular endotoxins. Particularly preferred examples of immunogenic epitopes are epitopes from herpes viruses such as the herpes virus 6 or pseudorabies virus (Lomniczi et al., J. Virol. 49 (1984), 970-979), in particular epitopes from the genes gB, gC or/and gD, or foot-and-mouth disease virus (FMDV), in particular epitopes from the gene sections which code for VP1, VP2 or/and VP3. The immunogenic epitopes can be selected such that they promote an antibody-mediated immune reaction or/and the production of a cellular immune reaction e.g. by stimulation of T cells. Examples of suitable allergenic epitopes are birch pollen allergens e.g. Bet v I (Ebner et al., J. Immunol. 150 (1993) 1047-1054). Antigenic epitope
Howorka Stefan
Kuen Beatrix
Lubitz Werner
Resch Stepanka
Sara Margit
Lubitz Werner
Minnifield N. M.
Rothwell Figg Ernst & Manbeck P.C.
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