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.

Description

[0001] The present invention concerns processes for the recombinant production of S-layer proteins and modified S-layer proteins in gram-negative host cells.

[0002] 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.

[0003] 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).

[0004] 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.

[0005] 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.

[0006] 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.

[0007] 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.degree. C., preferably 60.degree. C. in an aqueous low salt buffer (e.g. 0.2.times.SSC) (see also Sambrook et al. (1989), Molecular Cloning. A Laboratory Manual).

[0008] 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.

[0009] The E. coli strain pop2125 which was deposited on the 31.01.1996 at the "Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH", Mascheroder Weg 1b, D 38124 Braunschweig under the file number DSM 10509 is particularly preferred.

[0010] 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.

[0011] 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.

[0012] 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.

[0013] 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.

[0014] 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 epitopes are additionally particularly preferred which are able to bind and filter out endogenous or exogenous substances such as cytokines or toxins from serum or other body fluids. Such epitopes can include components of cytokine or toxin receptors or of antibodies against cytokines or toxins.

[0015] On the other hand the insertions can also code for enzymes. Preferred examples are enzymes for the synthesis of polyhydroxybutyric acid e.g. PHB synthase. Incorporation of PHB synthase into the S-layer can lead to the formation of a molecular spinning nozzle under suitable conditions when the substrate hydroxybutyric acid is provided. A further preferred example of an enzyme is bacterial luciferase. In this case when the enzyme substrate, an aldehyde, is supplied and O.sub.2 is present, a molecular laser can be obtained.

[0016] Insertions are likewise preferred which code for cytokines such as interleukins, interferones or tumour necrosis factors. These molecules can for example be used in combination with immunogenic epitopes to prepare vaccines.

[0017] Finally insertions are also preferred which code for antibody binding proteins such as protein A or protein G or for DNA-binding or/and metal-binding epitopes such as the leucine zipper, zinc finger etc.

[0018] Thus for the first time a cell is provided by the present invention which contains immobilized recombinant polypeptides in a native form e.g. active enzymes in the cytoplasm. In this manner 50,000-200,000 e.g. ca. 100,000 recombinant molecules can be immobilized per m.sup.2 recombinant S-layer. Up to 3000 m.sup.2 S-layer can be obtained per kg recombinant E. coli cells.

[0019] In the method according to the invention the nucleic acid coding for the S-layer protein is preferably used in operative linkage with a nucleic acid coding for a signal peptide of gram-positive bacteria i.e. the signal peptide-coding nucleic acid is located on the 5' side of the S-layer protein-coding nucleic acid. Surprisingly it was found that the presence of such signal peptide sequences, which are not cleaved in the gram-negative host cells used in the invention, can improve the stability of the S-layer structures. The nucleic acid coding for the signal peptide particularly preferably comprises (a) the signal peptide-coding section of the nucleotide sequence shown in SEQ ID NO. 1, (b) a nucleotide sequence corresponding to the sequence from (a) within the scope of the degeneracy of the genetic code or/and (c) a nucleotide sequence which is at least 80% and in particular at least 90% homologous to the sequences from (a) or/and (b).

[0020] Yet a further subject matter of the present invention is a nucleic acid which codes for a recombinant S-layer protein and is 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 signal peptide-coding-section (ii) a nucleic acid which comprises a nucleotide sequence corresponding to a 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 under stringent conditions with the nucleic acids from (i) or/and (ii).

[0021] The coding nucleotide sequence of the S-layer gene sbsA from B.stearothermophilus including the signal peptide-coding section is shown in SEQ ID NO. 1. The signal peptide-coding section extends from position 1 to 90 of the nucleotide sequence shown in SEQ ID NO. 1. The section coding for the mature SbsA polypeptide extends from position 91 to 3684.

[0022] The sbsA gene of B.stearothermophilus codes for a protein with a total of 1228 amino acids including an N-terminal signal peptide with 30 amino acids (SEQ ID NO. 2). 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 has a basic amino-terminal domain followed by a hydrophobic domain.

[0023] Sequence comparisons with other signal peptides indicate a certain homology to signal peptides of extracellular proteins in bacilli such as alkaline phosphatase and neutral phosphatase of B.amyloliquefaciens (Vasantha et al., J. Bacteriol. 159 (1984), 811-819) as well as with the signal peptides for the B.sphaericus gene 125 (Bowditch et al., J. Bacteriol. 171 (1989), 4178-4188) and the OWP gene of B.brevis (Tsuboi et al., J. Bacteriol. 168 (1986), 365-373).

[0024] A further subject matter of the present invention is a recombinant vector which contains at least one copy of a nucleic acid according to the invention. The vector is preferably replicatable in prokaryotes. The vector is particularly preferably a prokaryotic plasmid.

[0025] Yet a further subject matter of the present invention is a host cell which is transformed with a nucleic acid or a recombinant vector according to the present invention. The cell is preferably a gram-negative prokaryotic organism and most preferably an E. coli cell. The cell according to the invention can contain a recombinant S-layer structure in its interior. Methods for the transformation of cells with nucleic acids are general state of the art (cf. Sambrook et al., supra) and therefore do not need to be elucidated.

[0026] Yet a further subject matter of the present invention is a recombinant S-layer protein which contains at least one peptide insertion or/and polypeptide insertion within the amino acid sequence shown in SEQ ID NO. 2. Preferred examples of peptide insertions and polypeptide insertions have already been elucidated.

[0027] A recombinant S-layer structure can be assembled from recombinant S-layer protein molecules according to the invention which contain at least one recombinant S-layer protein according to the invention as a subunit. Furthermore it is preferred that the S-layer structure according to the invention also contains non-modified S-layer proteins as diluent molecules. The non-modified S-layer proteins are preferably present in a molar proportion of 10-99% relative to the total S-layer proteins.

[0028] The S-layer structure according to the invention can comprise several layers that are covalently linked together or by means of affinity binding. Covalent linkages can for example be introduced by insertions of cysteine residues and a subsequent formation of cystine bridges. Linkages by affinity binding comprise for example antibody-antigen, antibody-protein A or antibody-protein G or streptavidin-biotin interactions.

[0029] S-layer structures which contain recombinant S-layer proteins can optionally also be prepared in a carrier-bound form. For this the S-layer structure can be reassembled from individual units in the presence of a peptidoglycan carrier to for example produce peptidoglycan layers which are covered on one or on both sides with an S-layer structure. Another method of preparing carrier-bound S-layer structures is to produce an S-layer layer at an interface between two media e.g. water/air and to immobilize this layer on a solid phase e.g. a filter membrane (cf. e.g. Pum and Sleytr (1994), Thin Solid Films 244, 882-886; Kupcu et al., (1995), Biochim. Biophys. Acta 1235, 263-269).

[0030] The recombinant S-layer proteins and S-layer structures according to the invention are suitable for a multitude of applications. An application as a vaccine or adjuvant is particularly preferred in which case recombinant S-layer proteins are used which contain immunogenic epitopes of pathogens and/or endogenous immunostimulatory polypeptides such as cytokines. In this application it is not absolutely necessary to purify the recombinant S-layer proteins. Instead they can for example be used in combination with a bacterial ghost which optionally contains additional immunogenic epitopes in its membrane.

[0031] The preparation of suitable "bacterial ghosts" is described for example in the International Patent application PCT/EP91/00967 to which reference is herewith made. In this application modified bacteria are disclosed which are obtainable by transformation of a gram-negative bacterium with the gene of a lytically active membrane protein from bacteriophages, with the gene of a lytically active toxin release protein or with genes which contain partial sequences thereof which code for lytic proteins, culturing the bacterium, expression of this lysis gene and isolation of the resulting bacterial ghost from the culture medium.

[0032] A recombinant protein, which is obtainable by expression of a recombinant DNA in these gram-negative bacteria, can be bound to the membrane of these bacteria as described in the European Patent 0 516 655. This recombinant DNA comprises a first DNA sequence which codes for a hydrophobic, non-lytically active membrane-integrating protein domain which has an .alpha.-helical structure and is composed of 14-20 amino acids which can be flanked N- and C-terminally by 2-30 arbitrary amino acids in each case. A second DNA sequence is in operative linkage with this first DNA sequence which codes for a desired recombinant protein. Furthermore the gram-negative bacterium contains a third DNA sequence which is under a different control from the first and second DNA sequences and codes for a lytically active membrane protein from bacteriophages or a lytically active toxin release protein or for their lytically active components. So-called "bacterial ghosts" are obtained by expression and lysis of such recombinant gram-negative bacteria which contain an intact surface structure with immunogenic epitopes bound to the surface.

[0033] When these bacterial ghosts are combined with recombinant S-layers according to the invention vaccines and adjuvants can be produced which have particularly advantageous properties.

[0034] A further particularly preferred application for recombinant S-layer proteins and S-layer structures is their use as an enzyme reactor. Such an enzyme reactor can for example be formed by a cell which contains a recombinant S-layer structure according to the invention in its interior. On the other hand the enzyme reactor can also be formed from isolated and in vitro reassembled S-layer structures or combinations of various S-layer structures.

[0035] It was found that the gram-positive bacterium B.stearothermophilus PV72 has an additional S-layer protein in addition to SbsA which is subsequently denoted as SbsB (Sara and Sleytr (1994), J. Bacteriol. 176, 7182-7189). It was possible to isolate and characterize the sbsB gene by amplification using suitable nucleic acid primers. The coding nucleotide sequence of the S-layer gene sbsB from B.stearothermophilus including the signal peptide-coding section which extends from position 1 to 93 of the nucleic acid sequence is shown in SEQ ID NO.5. The amino acid sequence derived therefrom is shown in SEQ ID NO.6. The sbsB gene codes for a protein with a total of 921 amino acids including an N-terminal signal peptide with 31 amino acids.

[0036] One subject matter of the present invention is hence a nucleic acid which codes for an S-layer protein and is selected from

[0037] (i) a nucleic acid which comprises the nucleotide sequence from position 1 to 2763 shown in SEQ ID NO.5 optionally without the signal peptide-coding section,

[0038] (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

[0039] (iii) a nucleic acid which comprises a nucleotide sequence that hybridizes with the nucleic acids from (i) or/and (ii) under stringent conditions.

[0040] As in the case of the sbsA-gene, it is also possible to insert at least one nucleic acid insertion coding for a peptide or polypeptide into the sbsB gene within the region coding for the S-layer protein. With regard to preferred examples of insertions in the sbsB gene reference is made to the previous statements regarding the sbsA gene.

[0041] Yet a further subject matter of the present invention is a vector which contains at least one copy of an sbsB gene optionally containing an insertion. This vector can be replicated in eukaryotes, prokaryotes or in eukaryotes and prokaryotes. It can be a vector that can be integrated into the genome of the host cell or a vector which is present extrachromosomally. The vector according to the invention is preferably a plasmid in particular a prokaryotic plasmid.

[0042] Yet a further subject matter of the present invention is a host cell which is transformed with an sbsB gene wherein the sbsB gene optionally can contain an insertion. The host cell can be a eukaryotic as well as a prokaryotic cell. The cell is preferably a prokaryotic organism. Gram-positive organisms e.g. organisms of the genus bacillus as well as gram-negative organisms such as enterobacteria in particular E. coli are preferred. Methods for transforming eukaryotic and prokaryotic cells with nucleic acids are known and therefore do not need to be elucidated in detail.

[0043] The present invention also concerns an SbsB protein i.e. an S-layer protein which is coded by a nucleic acid as defined above. Recombinant SbsB proteins are particularly preferred which contain one or several peptide or/and polypeptide insertions within the sbsB sequence. The SbsB part of a polypeptide according to the invention particularly preferably has a homology of at least 80% and in particular of at least 90% to the amino acid sequence shown in SEQ ID NO.6.

[0044] A recombinant S-layer structure can also be assembled from the recombinant SbsB-S-layer protein molecules analogous to the recombinant SbsA-S-layer structure. In this structure the non-modified S-layer proteins are preferably present in a molar proportion of 10-99% relative to the total S-layer proteins.

[0045] The applications for the recombinant SbsB-S-layer proteins and S-layer structures according to the invention also correspond to the applications for SbsA mentioned above. In this connection its use as a vaccine or adjuvant or as an enzyme reactor is noteworthy.

[0046] Recombinant S-layer proteins are obtainable by a process in which

[0047] (a) a host cell is provided which contains a nucleic acid coding for an S-layer protein which contains a peptide-coding or polypeptide-coding insertion within the region coding for the S-layer protein,

[0048] (b) the host cell is cultured under conditions which lead to an expression of the nucleic acid and to production of the polypeptide coded by it and

[0049] (c) the resulting polypeptide is isolated from the host cell or from the culture medium.

[0050] In a first preferred embodiment of this process a recombinant SbsA-S-layer protein is prepared i.e. the nucleic acid coding for the recombinant S-layer protein is selected from

[0051] (i) a nucleic acid which comprises the nucleotide sequence from position 1 to 3684 shown in SEQ ID NO.1 optionally without the signal peptide-coding section,

[0052] (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

[0053] (iii) a nucleic acid which comprises a nucleotide sequence which hybridizes with the nucleic acids from (i) or/and (ii) under stringent conditions.

[0054] In a second preferred embodiment a recombinant SbsB-S-layer protein is prepared i.e. the nucleic acid coding for the recombinant S-layer protein is selected from

[0055] (i) a nucleic acid which comprises the nucleotide sequence from position 1 to 2763 shown in SEQ ID NO.5 optionally without the signal peptide-coding section,

[0056] (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

[0057] (iii) a nucleic acid which comprises a nucleotide sequence which hybridizes with the nucleic acids from (i) or/and (ii) under stringent conditions.

[0058] In addition to the recombinant SbsA and SbsB-S-layer proteins from B.stearothermophilus it is, however, also possible to prepare recombinant S-layer proteins from other organisms (cf. e.g. Peyret et al., (1993), supra).

[0059] The recombinant S-layer proteins can on the one hand be produced in a heterologous host cell i.e. in a host cell which originally contains no S-layer gene. Examples of such heterologous host cells are gram-negative prokaryotic organisms such as E. coli.

[0060] However, the heterologous expression of S-layer proteins can also take place in gram-positive prokaryotic organisms such as B. subtilis. For this integration vectors are preferably used which contain a native or/and a recombinant S-layer gene. When the native signal sequences are used the S-layer proteins are secreted into the culture supernatant.

[0061] However, it is often preferable to produce the recombinant S-layer proteins in homologous host cells i.e. host cells which originally contain a natural S-layer gene. In one embodiment of this homologous expression the recombinant S-layer gene is introduced into the host cell in such a way that the host cell is still able to express a further S-layer gene which codes for a non-modified S-layer protein. The non-modified S-layer protein is preferably capable of forming an S-layer structure that is compatible with the recombinant S-layer protein. An example of this embodiment of homologous expression is a B.stearothermophilus PV72 cell which contains intact natural sbsA genes or/and sbsB genes and is transformed with a plasmid which contains a recombinant S-layer-gene.

[0062] In a second embodiment the homologous expression can occur in a host cell in which the intact S-layer gene originally present has been inactivated. Consequently in this embodiment no further S-layer gene is expressed in the host cell which codes for a non-modified S-layer protein which is able to form a compatible S-layer structure with the recombinant S-layer protein. A specific example of such a host cell is a B.stearothermophilus PV72 cell in the genome of which a gene coding for a recombinant S-layer protein has been introduced, e.g. by homologous recombination, which replaces the original S-layer gene. A further example of such a host cell is a B.stearothermophilus cell in which the native S-layer gene has been inactivated e.g. by site-specific mutagenesis or/and homologous recombination and is transformed with a vector containing a recombinant S-layer gene.

[0063] Gram-positive prokaryotic organisms are usually used as host cells for the homologous expression of recombinant S-layer genes. B.stearothermophilus PV72 is particularly preferred as a host cell which can be cultured at a high temperature in a defined synthetic medium (Schuster et al., (1995), Biotechnol. and Bioeng. 48: 66-77).

[0064] The present invention is further elucidated by the following examples and figures.

[0065] SEQ ID NO.1 shows the complete nucleotide sequence of the coding section of the S-layer gene sbsA of B.stearothermophilus;

[0066] SEQ ID NO.2 shows the amino acid sequence derived therefrom;

[0067] SEQ ID NO.3 shows the nucleotide sequence of the primer T5-X;

[0068] SEQ ID NO.4 shows the nucleotide sequence of the primer E;

[0069] SEQ ID NO.5 shows the complete nucleotide sequence of the coding section of the S-layer gene sbsB of B.stearothermophilus;

[0070] SEQ ID NO.6 shows the amino acid sequence derived therefrom;

[0071] SEQ ID NO.7 shows the nucleotide sequence of a partial fragment of the streptavidin gene;

[0072] SEQ ID NO.8 shows the nucleotide sequence of the primer NIS 2AG;

[0073] SEQ ID NO.9 shows the nucleotide sequence of the primer LIS C3;

[0074] FIG. 1 shows a schematic representation of the sbsA PCR fragment used to prepare the recombinant vector pBK4;

[0075] FIG. 2 shows a schematic representation of peptide insertions in the amino acid sequence of the SbsA S-layer protein and

[0076] FIG. 3 shows a schematic representation of amino acid substitutions and amino acid insertions in recombinant S-layer proteins.

EXAMPLES

[0077] 1. Bacterial Strains, Media and Plasmids

[0078] Gram-positive bacteria of the strain Bacillus stearothermophilus PV72 were cultured at 58.degree. C. in SVIII medium (Bartelmus and Perschak, Z. Zuckerrind. 7 (1957), 276-281). Bacteria of the strain E. coli pop2135 (endA, thi, hsdr, malT, cI857, .lambda.pR, malPQ) were cultured in LB medium (Sambrook et al., (1989), supra). Ampicillin was added to the medium at a final concentration of 100 .mu.g/ml to select for transformants. The plasmid pPLcAT10 (.lambda.pL, bla, colE1) (Stanssens et al., Gene 36 (1985), 211-223) was used as the cloning vector.

[0079] 2. Manipulation of DNA Fragments

[0080] Restriction analysis of DNA, agarose gel electrophoresis and cloning of DNA fragments were carried out according to the standard methods described in Sambrook et al. (1989), supra.

[0081] Competent cells were transformed by electroporation using a Bio-Rad gene pulser (Bio-Rad Laboratories, Richmond, Calif. USA) according to the manufacturer's instructions.

[0082] Plasmid DNA was isolated by the method of Birnboim and Doly (Nucleic Acids Res. 7 (1979), 1513-1523). Chromosomal DNA was isolated according to the method described in Ausubel et al. (Current Protocols in Molecular Biology (1987), New York, John Wiley).

[0083] Restriction endonucleases and other enzymes were obtained from Boehringer Mannheim, New England Biolabs or Stratagene and used according to the manufacturer's instructions.

[0084] 3. DNA Sequencing

[0085] The DNA sequences of the 5' regions and the 3' regions (including the region coding for the signal sequence) of the gene sbsA in the vector pPLcAT10 were determined by the dideoxy chain termination method of Sanger et al. The primers used for sequencing were constructed on the basis of the already published sbsA sequence (Kuen et al. Gene 145 (1994), 115-120).

[0086] 4. PCR Amplification of sbsA

[0087] The PCR amplification of the sbsA gene was carried out in a reaction volume of 100 .mu.l in which 200 .mu.M deoxynucleotides, 1 U Pfu-polymerase (Stratagene), 1.times. Pfu-reaction buffer, 0.5 .mu.M of each oligonucleotide primer and 100 ng genomic DNA from B.stearothermophilus as a template were present. The amplification was carried out for 30 cycles in a thermocycler (Biomed thermocycler 60). Each cycle was composed of a denaturing step of 1.5 min at 95.degree. C., an annealing step of 1 min at 56.degree. C. and 1 min at 50.degree. C. as well as an extension step of 2 min at 72.degree. C.

[0088] The primer T5-X shown in the sequence protocol as SEQ ID NO.3 which flanks the 5' region of sbsA and contains an XbaI site and the primer E shown in the sequence protocol in SEQ ID NO.4 which flanks the 20 nucleotide upstream region of the transcription terminator of the sbsA sequence and contains a BamHI site were used as primers.

[0089] The products amplified by PCR were electrophoretically separated on a 0.8% agarose gel and purified for cloning using the system from Gene Clean (BIO101 La Jolla, Calif. USA).

[0090] 5. Cloning of the sbsA Gene into the Vector pPLcAT10

[0091] The sbsA gene obtained by PCR with a length of 3.79 kb was purified and cleaved with the restriction endonucleases XbaI and BamHI. The resulting XbaI-BamHI fragment was cloned into the corresponding restriction sites of the vector pPLcAT10 so that the sbsA gene was under transcriptional control of the pL promoter located upstream. The ATG start codon of the sbsA sequence was reconstructed by the cloning procedure. The cloned sbsA sequence contained the N-terminal signal sequence of sbsA and ended 20 nt after the transcription terminator. After ligation of the vector DNA with the sbsA fragment, the E. coli strain pop2135 was transformed by electrotransformation. The resulting clones were subjected to a DNA restriction analysis. A positive clone was sequenced in order to verify the correct sequence transitions at the 5' and 3' ends. This clone was named pBK4.

[0092] A schematic representation of the 3.79 kb XbaI sbsA fragment and its location in the multiple cloning site of the plasmid pBK4 is shown in FIG. 1 (abbreviations: tT: transcription terminator; ori: origin of the DNA replication; amp: ampicillin resistance gene).

[0093] 6. Recombinant Expression of the SbsA Gene in E. coli

[0094] E. coli pop2135/pBK4 cells were cultured at 28.degree. C. until an optical density OD.sub.600 of 0.3 was reached. Then the expression of sbsA was induced by increasing the culture temperature from 28.degree. C. to 42.degree. C. 1.5 ml aliquots were taken before and 1, 2, 3 and 5 hours after induction of the sbsA expression. E. coli pop2135/pPLcAT10 (cultured under the same conditions) and B.stearothermophilus PV72 were used as controls.

[0095] Culture supernatants and cell extracts from all samples were examined for the expression of S-layer proteins by SDS-PAGE and Western immunoblotting.

[0096] An additional strong protein band with the same molecular weight as the wild type SbsA protein was found in extracts from E. coli cells transformed with pBK4. No degradation products of SbsA itself were found in a period of up to 5 hours after induction of expression. Thus presumably the S-layer protein sbsA is stable in E. coli and is not degraded by proteases.

[0097] A densitometric determination of the relative amount of SbsA protein was carried out. At a time point of 4 hours after induction the sbsA protein was in a proportion of ca. 16% relative to the total cellular protein.

[0098] The SbsA protein produced in E. coli migrated in the SDS gel slightly more slowly than the natural SbsA protein from B.stearothermophilus. Experiments to determine the N-terminal amino acid sequence of the SbsA protein by Edman degradation were not successful due to a blocking of the N-terminus. Thus presumably the signal sequence was not cleaved in E. coli.

[0099] A Western blot analysis of total cell extracts and culture supernatants of E. coli/pBK4 also only yielded a single sbsA-specific protein band with a slightly higher molecular weight than wild type SbsA protein from stearothermophilus.

[0100] For the Western blot the proteins were transferred onto a nitrocellulose membrane and incubated with a polyclonal antiserum against SbsA from rabbits. The preparation of this antiserum is described in Egelseer et al. (J. Bacteriol. 177 (1995), 1444-1451). A conjugate of goat anti-rabbit IgG and alkaline phosphatase was used to detect bound SbsA-specific antibodies.

[0101] No SbsA protein could be detected from supernatants from E. coli cells transformed with pBK4 even after induction of sbsA gene expression. This shows that SbsA is not exported into the surrounding medium.

[0102] 7. Location and Organisation of the S-layer Protein SbsA in the Cytoplasm of E. coli

[0103] Cells of E. coli pop2135/pBK4 which were harvested from cultures 1, 2, 3 and 5 hours after induction of the S-layer protein expression were examined for the intracellular organisation of sbsA. Non-induced cells cultured at 28.degree. C. and cells of B.stearothermophilus PV72 were examined as controls.

[0104] For this whole cells of both organisms were fixed and embedded in detection resin according to the method of Messner et al. (Int. J.Syst.Bacteriol. 34 (1984), 202-210). Subsequently ultrathin sections of the embedded preparations were prepared and stained with uranyl acetate.

[0105] The cytoplasm of non-induced E. coli cells exhibited the typical granular structure which did not change even when the OD of the suspensions increased. Longitudinal sections of E. coli cells which were harvested 1 hour after induction of the S-layer protein expression exhibited parallel, leaf-like structures in the cytoplasm. From cross sections it was apparent that these structures have a concentric arrangement.

[0106] The amount of leaf-like structures considerably increased between 1 and 2 hours after induction of the sbsA expression and afterwards remained essentially constant.

[0107] The sbsA protein recombinantly produced in E. coli could also be detected by immunogold labelling with sbsA-specific antibodies. An ordered structure of the recombinantly produced SbsA protein was also found with this detection method.

[0108] It was clearly apparent from these morphological data that the SbsA protein did not aggregate to form irregular inclusion bodies but rather formed monomolecular S-layer crystals. A remarkable property of the SbsA-S-layer layers assembled in E. coli was the concentric arrangement at defined distances. The presence of the signal sequence did not interfere with correct assembly.

[0109] 8. Preparation of Recombinant sbsA-S-Layer Genes

[0110] 8.1 Insertion of a 6 bp Long DNA Sequence

[0111] A modified kanamycin cassette (1.3 kb) was used for the site-specific insertion mutagenesis of the sbsA gene which was isolated by cleavage of the plasmid pWJC3 (obtained from W. T. McAllister, New York) by SmaI. The cassette was ligated into five different blunt-ended restriction sites of the sbsA gene, i.e. into the NruI site at position bp 582 (pSL582), into the SnaBI site at position bp 917 (pSL917) and into each of the PvuII sites at positions bp 878 (pSL878), bp 2504 (pSL2504) and bp 2649 (pSL2649). After selection of kanamycin-resistant clones, the cassette was removed from the insertion site by cleavage with ApaI followed by a religation of the S-layer plasmid pBK4. The cutting out and religation procedure left an insertion of 6 bp CCCGGG (ApaI restriction site). The system of this linker insertion is shown schematically in FIG. 2.

[0112] The resulting recombinant S-layer genes code for modified sbsA proteins elongated by 2 amino acids.

[0113] The specific changes in the primary structure of the sbsA proteins are shown in FIG. 3. In the clone pSL582 the insertion led to the incorporation of glycine and proline between the amino acids 194 and 195 at the N-terminus of the SbsA protein. The amino acids alanine and arginine were inserted in the clone pSL917 between the amino acids 306 and 307. In the clone pSL2649 glycine and proline were inserted between the amino acids at positions 883 and 884. An insertion of alanine and proline between the amino acids 293 and 294 was obtained in the clone pSL878. Furthermore the alanine at position 293 was substituted by glycine. In the clone pSL2504 the amino acids alanine and proline were inserted between the amino acids 835 and 836 and the alanine at position 835 was replaced by glycine.

[0114] All clones obtained by insertion mutagenesis retained their ability to synthesise the S-layer protein.

[0115] In order to test the ability of the modified proteins to assemble into S-layer structures, ultrathin longitudinal sections of whole cells which had been cultured for 4 hours under inductive conditions were prepared according to the procedure described in section 7. It was found that the cytoplasm of all five clones is filled with parallel, leaf-like structures which follow the curve of the cell poles. There were no morphological differences of the cytoplasm in the 5 different clones examined. Exactly the same leaf-like structures were found as in the assembly of the wild type SbsA protein in E. coli (section 7).

[0116] 8.2 Insertion of a DNA Sequence Coding for Streptavidin

[0117] In order to examine whether the insertion of larger protein sequences into the SbsA protein can also be tolerated, a DNA fragment coding for a part of streptavidin (160 amino acids) provided with ApaI linkers (SEQ ID NO.7) was gene inserted into the ApaI restriction site of the sbsA clones pSL582, pSL878, pSL917 and pSL2649 prepared in the example on page 1. The streptavidin sequence was inserted in SL582 in the codon 197, in pSL878 between codon 295 and 296, in pSL917 in the codon 308 and 309 and in pSL2649 in the codon 886. It was possible to detect the expression of SbsA-streptavidin fusion proteins in all constructs by SDS-PAGE and immunoblots. It was found by EM analysis that a self assembly of the S-layer structure was possible in the fusion proteins containing insertions in the codon 197 and between the codons 295 and 296.

[0118] The SbsA-streptavidin fusion proteins can be isolated as monomers and reassembled to form homogeneous SbsA-streptavidin S-layers or mixed SbsA-streptavidin/SbsA-S-layers. They can be used to bind biotinylated substances as well as to determine the binding capacity of enzymes and other bound molecules.

[0119] 8.3 Insertion of a DNA Sequence Coding for BetvI

[0120] A DNA sequence coding for the open reading frame of BetvI (161 amino acids) the main pollen allergen of the birch (Ferreira et al., J. Biol. Chem. 268 (1993), 19574-19580) was inserted at the ApaI site into the sbsA clone pSL878. It was possible to detect the expression of an SbsA-BetvI fusion protein which contained an immunologically active BetvI domain.

[0121] The resulting fusion protein can be used for therapeutic or diagnostic purposes. Hence it can be attempted by administration of the fusion protein to convert a T.sub.H2-directed IgE antibody reaction into a T.sub.H1-mediated reaction against BetvI. In this manner it is possible to suppress the occurrence of symptoms of a pollen allergy. Furthermore SbsA-BetvI fusion proteins can be used to test for anti-BetvI antibody concentrations or/and to reduce high concentrations of anti-BetvI IgE.

[0122] 8.4 Insertion of a DNA Sequence Coding for a Pseudorabies Virus Antigen

[0123] The DNA sequence coding for the gB epitope SmaBB (255 amino acids) (nucleotides 489-1224 corresponding to the coordinates according to the EMBL-Seq: HEHSSGP2) from the pseudorabies virus was inserted into SSPI site of the sbsA gene after nt 3484 (between codon 1161 and 1162). It was possible to detect the expression of SbsA-SmaBB fusion proteins.

[0124] The fusion proteins can be used to test gB-specific immune reactions. A Western blot analysis using a monoclonal antibody which corresponds to the inserted sequence showed the immunological activity of the viral domain within the recombinant SbsA-SmaBB proteins.

[0125] 8.5 Insertion of a DNA Sequence Coding for the PHB Synthase (PhbC) from Alcaligenes Eutrophus H16

[0126] A regular arrangement of polypeptide structures with enzymatic activity on the surface of S-layers is an important goal in the production of immobilized enzymes within a living cell and in the case of the 590 amino acid long PHB synthase for the production of a molecular machine for biopolymer synthesis.

[0127] The phbC gene was isolated by PCR from the plasmid p4A (Janes et al., Molecular characterisation of the poly-.beta.-hydroxy-butyrate biosynthesis in Alcaligenes eutrophus H16. In: Novel Biodegradable Microbial Polymers (publisher Daves, E. A.), pp 175-190 (1990), Kluver, Dordrecht) as a 1770 nt long DNA fragment (corresponding to an open reading frame of 590 amino acids) and inserted into the ApaI cleavage site of the sbsA clone pSL878 to obtain the plasmid pSbsA-PhbC. It was possible to detect the expression of an SbsA-PhbC fusion protein of ca. 195 kD in an E. coli cell transformed with this plasmid. When two copies of the phbc gene were inserted one behind the other into the ApaI site of pSL878, it was possible to detect the expression of a fusion protein of ca. 260 kD.

[0128] For a functional test of the enzymatic activity of the SbsA-PhbC construct, the E. coli cells which contained the plasmid pSbsA-PhbC were co-transformed with the plasmid pUMS which contains the .beta.-ketothiolase (PhbA) and the acetoacetyl-CoA reductase (PhbB) from A. eutrophus (Kalousek et al., Genetic engineering of PHB-synthase from Alcaligenes eutrophus H16. In: Proceedings of the International Symposium on Bacterial Polyhydroxy-alkanoates, pp 426-427 (1993), publisher Schlegel H. G., Steinbuchel A. Goltze Press, Gottingen). The poly-.beta.-hydroxybutyrate formation in the co-transformed E. coli cells was detectable by staining with Sudan black, gas chromatography and electron microscopy. These findings show that the SbsA-PhbC construct is enzymatically active and represents a successful example of the immobilization of enzymes on intracellular S-layer matrices.

[0129] 8.6 Insertion of a DNA Sequence Coding for a Bacterial Luciferase Gene

[0130] A monocistronic LuxAB gene with a length of 2,070 nt which contains the fusion protein LuxAB composed of the two subunits LuxB and LuxB of the bacterial luciferase from Vibrio harveyi was isolated from the plasmid pT7-mut3 (Boylan et al., J. Biol. Chem. 264 (1989), 1915-1918) by PCR and inserted into the ApaI site of the clone pSL878 prepared in example 8.1 to obtain the plasmid pBK878-LuxAB. It was possible to detect the expression of an SbsA-PhbC fusion protein of ca. 207 kD in an E. coli cell transformed with this plasmid. The enzymatic activity of,the fusion protein was demonstrated by the method described in Boylan et al., Supra.

[0131] 9. Isolation and Characterization of the SbsB Gene

[0132] The basis for the isolation of the sbsB gene was the amino acid sequence of the N-terminus as well as the sequence of three internal peptides of the SbsB protein. Starting with these peptide sequences, degenerate oligonucleotide primers were constructed and used for the PCR. In this manner a 1076 bp long PCR fragment from the chromosomal DNA of B.stearothermophilus was amplified, cloned and sequenced (corresponding to position 100-1176 of the sequence shown in SEQ ID No.5).

[0133] The method of inverse PCR was used to amplify the sections on the 5' side and 3' side of the sbsB gene and stepwise overlapping DNA fragments were obtained with the aid of various primer combinations and sequenced.

[0134] The primer NIS 2AG shown in the sequence protocol as SEQ ID NO.8 which contains the 5' region of sbsB as well as the primer LIS C3 shown in the. sequence protocol of SEQ ID NO.9 which contains the 3' region of sbsB were used as primers to amplify the complete sbsB gene.

[0135] The PCR fragment obtained in this manner which contains the nucleotide sequence shown in SEQ ID NO.5 with 5' and 3' BamHI restriction cleavage sites was cloned as described in example 5 into the vector pPLcAT10 in which the expression takes place under the control of the lambda PL promoter.

[0136] Furthermore the sbsB-PCR fragment with the 5' side EcoRI and 3' side BamHi cleavage site were cloned into the vector pUC18 in which the expression took place under the control of the lac promoter.

[0137] The detection of the sbsB expression was carried out as described in examples 6 and 7 by SDS gel electrophoresis and electron microscopy.

[0138] 10. Preparation of Recombinant SbsB-S-layer Genes

[0139] Recombinant sbsB genes were prepared analogously to the methods described in example 8.

[0140] Thus in accordance with the method described in example 8.1, a 6 nt long DNA sequence containing an ApaI restriction cleavage site was introduced at various positions into the sbsB-layer gene. The recombinant sbsB clones pAK407, pAK481 and pAK1582 with ApaI cleavage sites at nt 407 (codon 136), 481 (codon 161/162) and 1582 (codon 528/529) were obtained in this manner. These clones obtained by insertion mutagenesis retained their ability to synthesize the S-layer protein and form S-layer structures.

[0141] Analogously to the method described in example 8.2, a DNA fragment coding for streptavidin was inserted into the ApaI restriction sites of the sbsB clones pAK407 and pAK481.

[0142] Analogously to example 8.4, a DNA sequence coding for the gB epitope SmaBB was inserted into the ApaI cleavage sites of the sbsB clones pAK481 and pAK1582. It was possible to detect the expression of sbsB-SmaB fusion proteins of ca. 130 kD in the E. coli cells transformed with the resulting recombinant plasmids. When two copies of the SmaBB epitopes were inserted one behind the other into the ApaI cleavage site of pAK481 it was possible to detect the expression of a fusion protein of ca. 157 kD. The SmaBB domains of the fusion proteins were recognized by specific antibodies.

[0143] Analogously to example 8.6 it was possible to detect the expression of a 175 kD SbsB-LuxAB fusion protein when the LuxAB sequence was inserted into the ApaI cleavage site of pAK407.

[0144] 11. Heterologous Expression of sbsA and sbsB in Bacillus subtilis

[0145] The integration vector pX (Kim, L., Mogk, A. and Schumann W., Gene 181 (1996), 71-76: A xylose-inducible Bacillus subtilis integration vector and its application) was used for the heterologous expression of sbsA and sbsB in B. subtilis. The S-layer genes in the resulting recombinant expression vectors are under the transcriptional control of the xyl promoter. Transformants of B.subtilis containing an S-layer gene integrated in the chromosome exhibited an expression of large amounts of S-layer proteins in the supernatant of the cells which was inducible by addition of xylose to the growth medium. This shows that the signal sequences of sbsA and sbsB are recognized by the B. subtilis cell.

[0146] In an analogous manner it was possible to achieve a heterologous expression of recombinant sbsA and sbsB layer genes in B. subtilis.

Sequence CWU 1

1

10 1 3687 DNA Bacillus stearothermophilus CDS (1)..(3684) sig_peptide (1)..(90) mat_peptide (91)..(3684) 1 atg gat agg aaa aaa gct gtg aaa cta gca aca gca agt gct att gca 48 Met Asp Arg Lys Lys Ala Val Lys Leu Ala Thr Ala Ser Ala Ile Ala -30 -25 -20 -15 gca agt gca ttt gtc gct gca aat cca aac gct tct gaa gcg gct aca 96 Ala Ser Ala Phe Val Ala Ala Asn Pro Asn Ala Ser Glu Ala Ala Thr -10 -5 -1 1 gat gta gca aca gta gta agc caa gca aaa gca cag ttc aaa aaa gca 144 Asp Val Ala Thr Val Val Ser Gln Ala Lys Ala Gln Phe Lys Lys Ala 5 10 15 tac tat act tac agc cat aca gta acg gaa act ggt gaa ttc cca aac 192 Tyr Tyr Thr Tyr Ser His Thr Val Thr Glu Thr Gly Glu Phe Pro Asn 20 25 30 att aac gat gta tat gct gaa tac aac aaa gcg aaa aaa cga tac cgt 240 Ile Asn Asp Val Tyr Ala Glu Tyr Asn Lys Ala Lys Lys Arg Tyr Arg 35 40 45 50 gat gcg gta gca tta gtg aat aaa gca ggt ggc gcg aaa aaa gac gct 288 Asp Ala Val Ala Leu Val Asn Lys Ala Gly Gly Ala Lys Lys Asp Ala 55 60 65 tac tta gct gat tta caa aaa gaa tat gaa act tac gtt ttc aaa gca 336 Tyr Leu Ala Asp Leu Gln Lys Glu Tyr Glu Thr Tyr Val Phe Lys Ala 70 75 80 aac cct aaa tct ggc gaa gct cgt gta gca act tac atc gat gct tac 384 Asn Pro Lys Ser Gly Glu Ala Arg Val Ala Thr Tyr Ile Asp Ala Tyr 85 90 95 aac tat gca aca aaa tta gac gaa atg cgc caa gag cta gag gct gct 432 Asn Tyr Ala Thr Lys Leu Asp Glu Met Arg Gln Glu Leu Glu Ala Ala 100 105 110 gtt caa gca aaa gat tta gaa aaa gca gaa caa tac tat cac aaa att 480 Val Gln Ala Lys Asp Leu Glu Lys Ala Glu Gln Tyr Tyr His Lys Ile 115 120 125 130 cct tat gaa att aaa act cgc aca gtc att tta gat cgc gta tat ggt 528 Pro Tyr Glu Ile Lys Thr Arg Thr Val Ile Leu Asp Arg Val Tyr Gly 135 140 145 aaa aca act cgt gat tta ctt cgc tct aca ttt aaa gca aaa gca caa 576 Lys Thr Thr Arg Asp Leu Leu Arg Ser Thr Phe Lys Ala Lys Ala Gln 150 155 160 gaa ctt cgc gac agc tta att tat gat att acc gtt gca atg aaa gcg 624 Glu Leu Arg Asp Ser Leu Ile Tyr Asp Ile Thr Val Ala Met Lys Ala 165 170 175 cgc gaa gta caa gac gct gtg aaa gca ggc aat tta gac aaa gct aaa 672 Arg Glu Val Gln Asp Ala Val Lys Ala Gly Asn Leu Asp Lys Ala Lys 180 185 190 gct gct gtt gat caa atc aat caa tac tta cca aaa gta aca gat gct 720 Ala Ala Val Asp Gln Ile Asn Gln Tyr Leu Pro Lys Val Thr Asp Ala 195 200 205 210 ttc aaa act gaa cta aca gaa gta gcg aaa aaa gca tta gat gca gat 768 Phe Lys Thr Glu Leu Thr Glu Val Ala Lys Lys Ala Leu Asp Ala Asp 215 220 225 gaa gct gcg ctt act cca aaa gtt gaa agt gta agt gcg att aac act 816 Glu Ala Ala Leu Thr Pro Lys Val Glu Ser Val Ser Ala Ile Asn Thr 230 235 240 caa aac aaa gct gtt gaa tta aca gca gta cca gtg aac gga aca cta 864 Gln Asn Lys Ala Val Glu Leu Thr Ala Val Pro Val Asn Gly Thr Leu 245 250 255 aaa tta caa ctt tca gct gct gca aat gaa gat aca gta aac gta aat 912 Lys Leu Gln Leu Ser Ala Ala Ala Asn Glu Asp Thr Val Asn Val Asn 260 265 270 act gta cgt atc tat aaa gtg gac ggt aac att cca ttt gcc ctt aat 960 Thr Val Arg Ile Tyr Lys Val Asp Gly Asn Ile Pro Phe Ala Leu Asn 275 280 285 290 acg gca gat gtt tct tta tct aca gac gga aaa act atc act gtg gat 1008 Thr Ala Asp Val Ser Leu Ser Thr Asp Gly Lys Thr Ile Thr Val Asp 295 300 305 gct tca act cca ttc gaa aat aat acg gag tat aaa gta gta gtt aaa 1056 Ala Ser Thr Pro Phe Glu Asn Asn Thr Glu Tyr Lys Val Val Val Lys 310 315 320 ggt att aaa gac aaa aat ggc aaa gaa ttt aaa gaa gat gca ttc act 1104 Gly Ile Lys Asp Lys Asn Gly Lys Glu Phe Lys Glu Asp Ala Phe Thr 325 330 335 ttc aag ctt cga aat gat gct gta gtt act caa gtg ttt gga act aat 1152 Phe Lys Leu Arg Asn Asp Ala Val Val Thr Gln Val Phe Gly Thr Asn 340 345 350 gta aca aac aac act tct gta aac tta gca gca ggt act ttc gac act 1200 Val Thr Asn Asn Thr Ser Val Asn Leu Ala Ala Gly Thr Phe Asp Thr 355 360 365 370 gac gat act tta aca gta gta ttt gat aag ttg tta gca cct gaa act 1248 Asp Asp Thr Leu Thr Val Val Phe Asp Lys Leu Leu Ala Pro Glu Thr 375 380 385 gta aac agc tcg aac gtt act att aca gat gtt gaa act gga aaa cgc 1296 Val Asn Ser Ser Asn Val Thr Ile Thr Asp Val Glu Thr Gly Lys Arg 390 395 400 att cca gta att gca tct act tct ggt tct aca att act att acg tta 1344 Ile Pro Val Ile Ala Ser Thr Ser Gly Ser Thr Ile Thr Ile Thr Leu 405 410 415 aaa gaa gcg tta gta act ggt aaa caa tat aaa ctt gct atc aat aat 1392 Lys Glu Ala Leu Val Thr Gly Lys Gln Tyr Lys Leu Ala Ile Asn Asn 420 425 430 gtt aaa aca tta act ggt tac aat gca gaa gct tac gag tta gtg ttc 1440 Val Lys Thr Leu Thr Gly Tyr Asn Ala Glu Ala Tyr Glu Leu Val Phe 435 440 445 450 act gca aac gca tca gca cca act gtt gct acc gct cct act act tta 1488 Thr Ala Asn Ala Ser Ala Pro Thr Val Ala Thr Ala Pro Thr Thr Leu 455 460 465 ggt ggt aca act tta tct act ggt tct ctt aca aca aat gtt tgg ggt 1536 Gly Gly Thr Thr Leu Ser Thr Gly Ser Leu Thr Thr Asn Val Trp Gly 470 475 480 aaa ttg gct ggt ggt gtg aat gaa gct gga act tat tat cct ggt ctt 1584 Lys Leu Ala Gly Gly Val Asn Glu Ala Gly Thr Tyr Tyr Pro Gly Leu 485 490 495 caa ttc aca aca acg ttt gct act aag tta gac gaa tct act tta gct 1632 Gln Phe Thr Thr Thr Phe Ala Thr Lys Leu Asp Glu Ser Thr Leu Ala 500 505 510 gat aac ttt gta tta gtt gaa aaa gaa tct ggt aca gtt gtt gct tct 1680 Asp Asn Phe Val Leu Val Glu Lys Glu Ser Gly Thr Val Val Ala Ser 515 520 525 530 gaa cta aaa tat aat gca gac gct aaa atg gta act tta gtg cca aaa 1728 Glu Leu Lys Tyr Asn Ala Asp Ala Lys Met Val Thr Leu Val Pro Lys 535 540 545 gcg gac ctt aaa gaa aat aca atc tat caa atc aaa att aaa aaa ggc 1776 Ala Asp Leu Lys Glu Asn Thr Ile Tyr Gln Ile Lys Ile Lys Lys Gly 550 555 560 ttg aag tcc gat aaa ggt att gaa tta ggc act gtt aac gag aaa aca 1824 Leu Lys Ser Asp Lys Gly Ile Glu Leu Gly Thr Val Asn Glu Lys Thr 565 570 575 tat gag ttc aaa act caa gac tta act gct cct aca gtt att agc gta 1872 Tyr Glu Phe Lys Thr Gln Asp Leu Thr Ala Pro Thr Val Ile Ser Val 580 585 590 acg tct aaa aat ggc gac gct gga tta aaa gta act gaa gct caa gaa 1920 Thr Ser Lys Asn Gly Asp Ala Gly Leu Lys Val Thr Glu Ala Gln Glu 595 600 605 610 ttt act gtg aag ttc tca gag aat tta aat aca ttt aat gct aca acc 1968 Phe Thr Val Lys Phe Ser Glu Asn Leu Asn Thr Phe Asn Ala Thr Thr 615 620 625 gtt tcg ggt agc aca atc aca tac ggt caa gtt gct gta gta aaa gcg 2016 Val Ser Gly Ser Thr Ile Thr Tyr Gly Gln Val Ala Val Val Lys Ala 630 635 640 ggt gca aac tta tct gct ctt aca gca agt gac atc att cca gct agt 2064 Gly Ala Asn Leu Ser Ala Leu Thr Ala Ser Asp Ile Ile Pro Ala Ser 645 650 655 gtt gaa gcg gtt act ggt caa gat gga aca tac aaa gtg aaa gtt gct 2112 Val Glu Ala Val Thr Gly Gln Asp Gly Thr Tyr Lys Val Lys Val Ala 660 665 670 gct aac caa tta gaa cgt aac caa ggg tac aaa tta gta gtg ttc ggt 2160 Ala Asn Gln Leu Glu Arg Asn Gln Gly Tyr Lys Leu Val Val Phe Gly 675 680 685 690 aaa ggt gca aca gct cct gtt aaa gat gct gca aat gca aat act tta 2208 Lys Gly Ala Thr Ala Pro Val Lys Asp Ala Ala Asn Ala Asn Thr Leu 695 700 705 gca act aac tat atc tat aca ttt aca act gaa ggt caa gac gta aca 2256 Ala Thr Asn Tyr Ile Tyr Thr Phe Thr Thr Glu Gly Gln Asp Val Thr 710 715 720 gca cca acg gtt aca aaa gta ttc aaa ggt gat tct tta aaa gac gct 2304 Ala Pro Thr Val Thr Lys Val Phe Lys Gly Asp Ser Leu Lys Asp Ala 725 730 735 gat gca gtt act aca ctt acg aac gtt gat gca ggt caa aaa ttc act 2352 Asp Ala Val Thr Thr Leu Thr Asn Val Asp Ala Gly Gln Lys Phe Thr 740 745 750 atc caa ttt agc gaa gaa tta aaa act tct agt ggt tct tta gtg ggt 2400 Ile Gln Phe Ser Glu Glu Leu Lys Thr Ser Ser Gly Ser Leu Val Gly 755 760 765 770 ggc aaa gta act gtc gag aaa tta aca aac aac gga tgg gta gat gct 2448 Gly Lys Val Thr Val Glu Lys Leu Thr Asn Asn Gly Trp Val Asp Ala 775 780 785 ggt act gga aca act gta tca gtt gct cct aag aca gat gca aat ggt 2496 Gly Thr Gly Thr Thr Val Ser Val Ala Pro Lys Thr Asp Ala Asn Gly 790 795 800 aaa gta aca gct gct gtg gtt aca tta act ggt ctt gac aat aac gac 2544 Lys Val Thr Ala Ala Val Val Thr Leu Thr Gly Leu Asp Asn Asn Asp 805 810 815 aaa gat gcg aaa ttg cgt ctg gta gta gat aag tct tct act gat gga 2592 Lys Asp Ala Lys Leu Arg Leu Val Val Asp Lys Ser Ser Thr Asp Gly 820 825 830 att gct gat gta gct ggt aat gta att aag gaa aaa gat att tta att 2640 Ile Ala Asp Val Ala Gly Asn Val Ile Lys Glu Lys Asp Ile Leu Ile 835 840 845 850 cgt tac aac agc tgg aga cac act gta gct tct gtg aaa gct gct gct 2688 Arg Tyr Asn Ser Trp Arg His Thr Val Ala Ser Val Lys Ala Ala Ala 855 860 865 gac aaa gat ggt caa aac gct tct gct gca ttc cca aca agc act gca 2736 Asp Lys Asp Gly Gln Asn Ala Ser Ala Ala Phe Pro Thr Ser Thr Ala 870 875 880 att gat aca act aag agc tta tta gtt gaa ttc aat gaa act gat tta 2784 Ile Asp Thr Thr Lys Ser Leu Leu Val Glu Phe Asn Glu Thr Asp Leu 885 890 895 gcg gaa gtt aaa cct gag aac atc gtt gtt aaa gat gca gca ggt aat 2832 Ala Glu Val Lys Pro Glu Asn Ile Val Val Lys Asp Ala Ala Gly Asn 900 905 910 gcg gta gct ggt act gta aca gca tta gac ggt tct aca aat aaa ttt 2880 Ala Val Ala Gly Thr Val Thr Ala Leu Asp Gly Ser Thr Asn Lys Phe 915 920 925 930 gta ttc act cca tct caa gaa tta aaa gct ggt aca gtt tac tct gta 2928 Val Phe Thr Pro Ser Gln Glu Leu Lys Ala Gly Thr Val Tyr Ser Val 935 940 945 aca att gac ggt gtg aga gat aaa gta ggt aac aca atc tct aaa tac 2976 Thr Ile Asp Gly Val Arg Asp Lys Val Gly Asn Thr Ile Ser Lys Tyr 950 955 960 att act tcg ttc aag act gta tct gcg aat cca acg tta tct tca atc 3024 Ile Thr Ser Phe Lys Thr Val Ser Ala Asn Pro Thr Leu Ser Ser Ile 965 970 975 agc att gct gac ggt gca gtt aac gtt gac cgt tct aaa aca att aca 3072 Ser Ile Ala Asp Gly Ala Val Asn Val Asp Arg Ser Lys Thr Ile Thr 980 985 990 att gaa ttc agc gat tca gtt cca aac cca aca atc act ctt aag aag 3120 Ile Glu Phe Ser Asp Ser Val Pro Asn Pro Thr Ile Thr Leu Lys Lys 995 1000 1005 1010 gct gac gga act tca ttt act aat tac act tta gta aat gta aat aat 3168 Ala Asp Gly Thr Ser Phe Thr Asn Tyr Thr Leu Val Asn Val Asn Asn 1015 1020 1025 gaa aat aaa aca tac aaa att gta ttc cac aaa ggt gta aca ctt gac 3216 Glu Asn Lys Thr Tyr Lys Ile Val Phe His Lys Gly Val Thr Leu Asp 1030 1035 1040 gag ttt act caa tat gag tta gca gtt tca aaa gat ttt caa act ggt 3264 Glu Phe Thr Gln Tyr Glu Leu Ala Val Ser Lys Asp Phe Gln Thr Gly 1045 1050 1055 act gat att gat agc aaa gtt aca ttc atc aca ggt tct gtt gct act 3312 Thr Asp Ile Asp Ser Lys Val Thr Phe Ile Thr Gly Ser Val Ala Thr 1060 1065 1070 gac gaa gta aaa cct gct cta gta ggc gtt ggt tca tgg aat gga aca 3360 Asp Glu Val Lys Pro Ala Leu Val Gly Val Gly Ser Trp Asn Gly Thr 1075 1080 1085 1090 agc tat act cag gat gct gca gca aca cga ctt cgg tct gta gct gac 3408 Ser Tyr Thr Gln Asp Ala Ala Ala Thr Arg Leu Arg Ser Val Ala Asp 1095 1100 1105 ttc gtt gcg gag cca gtt gcc ctt caa ttc tca gaa ggt atc gat tta 3456 Phe Val Ala Glu Pro Val Ala Leu Gln Phe Ser Glu Gly Ile Asp Leu 1110 1115 1120 acg aat gca act gtg aca gta aca aat att act gat gat aaa act gtt 3504 Thr Asn Ala Thr Val Thr Val Thr Asn Ile Thr Asp Asp Lys Thr Val 1125 1130 1135 gaa gtt att tca aaa gag agt gta gac gca gac cat gat gca ggt gct 3552 Glu Val Ile Ser Lys Glu Ser Val Asp Ala Asp His Asp Ala Gly Ala 1140 1145 1150 act aag gag aca tta gta att aac aca gtt act cct tta gta ctt gat 3600 Thr Lys Glu Thr Leu Val Ile Asn Thr Val Thr Pro Leu Val Leu Asp 1155 1160 1165 1170 aac agc aag act tat aag att gtt gta agt gga gtt aaa gat gca gca 3648 Asn Ser Lys Thr Tyr Lys Ile Val Val Ser Gly Val Lys Asp Ala Ala 1175 1180 1185 ggt aat gtt gca gat act att aca ttc tat att aag taa 3687 Gly Asn Val Ala Asp Thr Ile Thr Phe Tyr Ile Lys 1190 1195 2 1228 PRT Bacillus stearothermophilus 2 Met Asp Arg Lys Lys Ala Val Lys Leu Ala Thr Ala Ser Ala Ile Ala -30 -25 -20 -15 Ala Ser Ala Phe Val Ala Ala Asn Pro Asn Ala Ser Glu Ala Ala Thr -10 -5 -1 1 Asp Val Ala Thr Val Val Ser Gln Ala Lys Ala Gln Phe Lys Lys Ala 5 10 15 Tyr Tyr Thr Tyr Ser His Thr Val Thr Glu Thr Gly Glu Phe Pro Asn 20 25 30 Ile Asn Asp Val Tyr Ala Glu Tyr Asn Lys Ala Lys Lys Arg Tyr Arg 35 40 45 50 Asp Ala Val Ala Leu Val Asn Lys Ala Gly Gly Ala Lys Lys Asp Ala 55 60 65 Tyr Leu Ala Asp Leu Gln Lys Glu Tyr Glu Thr Tyr Val Phe Lys Ala 70 75 80 Asn Pro Lys Ser Gly Glu Ala Arg Val Ala Thr Tyr Ile Asp Ala Tyr 85 90 95 Asn Tyr Ala Thr Lys Leu Asp Glu Met Arg Gln Glu Leu Glu Ala Ala 100 105 110 Val Gln Ala Lys Asp Leu Glu Lys Ala Glu Gln Tyr Tyr His Lys Ile 115 120 125 130 Pro Tyr Glu Ile Lys Thr Arg Thr Val Ile Leu Asp Arg Val Tyr Gly 135 140 145 Lys Thr Thr Arg Asp Leu Leu Arg Ser Thr Phe Lys Ala Lys Ala Gln 150 155 160 Glu Leu Arg Asp Ser Leu Ile Tyr Asp Ile Thr Val Ala Met Lys Ala 165 170 175 Arg Glu Val Gln Asp Ala Val Lys Ala Gly Asn Leu Asp Lys Ala Lys 180 185 190 Ala Ala Val Asp Gln Ile Asn Gln Tyr Leu Pro Lys Val Thr Asp Ala 195 200 205 210 Phe Lys Thr Glu Leu Thr Glu Val Ala Lys Lys Ala Leu Asp Ala Asp 215 220 225 Glu Ala Ala Leu Thr Pro Lys Val Glu Ser Val Ser Ala Ile Asn Thr 230 235 240 Gln Asn Lys Ala Val Glu Leu Thr Ala Val Pro Val Asn Gly Thr Leu 245 250 255 Lys Leu Gln Leu Ser Ala Ala Ala Asn Glu Asp Thr Val Asn Val Asn 260 265 270 Thr Val Arg Ile Tyr Lys Val Asp Gly Asn Ile Pro Phe Ala Leu Asn 275 280 285 290 Thr Ala Asp Val Ser Leu Ser Thr Asp Gly Lys Thr Ile Thr Val Asp 295 300 305 Ala Ser Thr Pro Phe Glu Asn Asn Thr Glu Tyr Lys Val Val Val Lys 310 315 320 Gly Ile Lys Asp Lys Asn Gly Lys Glu Phe Lys Glu Asp Ala Phe Thr 325 330 335 Phe Lys Leu Arg Asn Asp Ala Val Val Thr Gln Val Phe Gly Thr Asn 340 345 350 Val Thr Asn Asn Thr Ser Val Asn Leu Ala Ala Gly Thr Phe Asp Thr 355 360 365 370 Asp Asp Thr Leu Thr Val Val Phe Asp Lys Leu Leu Ala Pro Glu Thr 375 380 385 Val Asn Ser Ser Asn Val Thr Ile Thr Asp Val Glu Thr Gly Lys Arg 390 395 400 Ile Pro Val Ile Ala Ser Thr Ser Gly Ser Thr Ile Thr Ile Thr Leu 405

410 415 Lys Glu Ala Leu Val Thr Gly Lys Gln Tyr Lys Leu Ala Ile Asn Asn 420 425 430 Val Lys Thr Leu Thr Gly Tyr Asn Ala Glu Ala Tyr Glu Leu Val Phe 435 440 445 450 Thr Ala Asn Ala Ser Ala Pro Thr Val Ala Thr Ala Pro Thr Thr Leu 455 460 465 Gly Gly Thr Thr Leu Ser Thr Gly Ser Leu Thr Thr Asn Val Trp Gly 470 475 480 Lys Leu Ala Gly Gly Val Asn Glu Ala Gly Thr Tyr Tyr Pro Gly Leu 485 490 495 Gln Phe Thr Thr Thr Phe Ala Thr Lys Leu Asp Glu Ser Thr Leu Ala 500 505 510 Asp Asn Phe Val Leu Val Glu Lys Glu Ser Gly Thr Val Val Ala Ser 515 520 525 530 Glu Leu Lys Tyr Asn Ala Asp Ala Lys Met Val Thr Leu Val Pro Lys 535 540 545 Ala Asp Leu Lys Glu Asn Thr Ile Tyr Gln Ile Lys Ile Lys Lys Gly 550 555 560 Leu Lys Ser Asp Lys Gly Ile Glu Leu Gly Thr Val Asn Glu Lys Thr 565 570 575 Tyr Glu Phe Lys Thr Gln Asp Leu Thr Ala Pro Thr Val Ile Ser Val 580 585 590 Thr Ser Lys Asn Gly Asp Ala Gly Leu Lys Val Thr Glu Ala Gln Glu 595 600 605 610 Phe Thr Val Lys Phe Ser Glu Asn Leu Asn Thr Phe Asn Ala Thr Thr 615 620 625 Val Ser Gly Ser Thr Ile Thr Tyr Gly Gln Val Ala Val Val Lys Ala 630 635 640 Gly Ala Asn Leu Ser Ala Leu Thr Ala Ser Asp Ile Ile Pro Ala Ser 645 650 655 Val Glu Ala Val Thr Gly Gln Asp Gly Thr Tyr Lys Val Lys Val Ala 660 665 670 Ala Asn Gln Leu Glu Arg Asn Gln Gly Tyr Lys Leu Val Val Phe Gly 675 680 685 690 Lys Gly Ala Thr Ala Pro Val Lys Asp Ala Ala Asn Ala Asn Thr Leu 695 700 705 Ala Thr Asn Tyr Ile Tyr Thr Phe Thr Thr Glu Gly Gln Asp Val Thr 710 715 720 Ala Pro Thr Val Thr Lys Val Phe Lys Gly Asp Ser Leu Lys Asp Ala 725 730 735 Asp Ala Val Thr Thr Leu Thr Asn Val Asp Ala Gly Gln Lys Phe Thr 740 745 750 Ile Gln Phe Ser Glu Glu Leu Lys Thr Ser Ser Gly Ser Leu Val Gly 755 760 765 770 Gly Lys Val Thr Val Glu Lys Leu Thr Asn Asn Gly Trp Val Asp Ala 775 780 785 Gly Thr Gly Thr Thr Val Ser Val Ala Pro Lys Thr Asp Ala Asn Gly 790 795 800 Lys Val Thr Ala Ala Val Val Thr Leu Thr Gly Leu Asp Asn Asn Asp 805 810 815 Lys Asp Ala Lys Leu Arg Leu Val Val Asp Lys Ser Ser Thr Asp Gly 820 825 830 Ile Ala Asp Val Ala Gly Asn Val Ile Lys Glu Lys Asp Ile Leu Ile 835 840 845 850 Arg Tyr Asn Ser Trp Arg His Thr Val Ala Ser Val Lys Ala Ala Ala 855 860 865 Asp Lys Asp Gly Gln Asn Ala Ser Ala Ala Phe Pro Thr Ser Thr Ala 870 875 880 Ile Asp Thr Thr Lys Ser Leu Leu Val Glu Phe Asn Glu Thr Asp Leu 885 890 895 Ala Glu Val Lys Pro Glu Asn Ile Val Val Lys Asp Ala Ala Gly Asn 900 905 910 Ala Val Ala Gly Thr Val Thr Ala Leu Asp Gly Ser Thr Asn Lys Phe 915 920 925 930 Val Phe Thr Pro Ser Gln Glu Leu Lys Ala Gly Thr Val Tyr Ser Val 935 940 945 Thr Ile Asp Gly Val Arg Asp Lys Val Gly Asn Thr Ile Ser Lys Tyr 950 955 960 Ile Thr Ser Phe Lys Thr Val Ser Ala Asn Pro Thr Leu Ser Ser Ile 965 970 975 Ser Ile Ala Asp Gly Ala Val Asn Val Asp Arg Ser Lys Thr Ile Thr 980 985 990 Ile Glu Phe Ser Asp Ser Val Pro Asn Pro Thr Ile Thr Leu Lys Lys 995 1000 1005 1010 Ala Asp Gly Thr Ser Phe Thr Asn Tyr Thr Leu Val Asn Val Asn Asn 1015 1020 1025 Glu Asn Lys Thr Tyr Lys Ile Val Phe His Lys Gly Val Thr Leu Asp 1030 1035 1040 Glu Phe Thr Gln Tyr Glu Leu Ala Val Ser Lys Asp Phe Gln Thr Gly 1045 1050 1055 Thr Asp Ile Asp Ser Lys Val Thr Phe Ile Thr Gly Ser Val Ala Thr 1060 1065 1070 Asp Glu Val Lys Pro Ala Leu Val Gly Val Gly Ser Trp Asn Gly Thr 1075 1080 1085 1090 Ser Tyr Thr Gln Asp Ala Ala Ala Thr Arg Leu Arg Ser Val Ala Asp 1095 1100 1105 Phe Val Ala Glu Pro Val Ala Leu Gln Phe Ser Glu Gly Ile Asp Leu 1110 1115 1120 Thr Asn Ala Thr Val Thr Val Thr Asn Ile Thr Asp Asp Lys Thr Val 1125 1130 1135 Glu Val Ile Ser Lys Glu Ser Val Asp Ala Asp His Asp Ala Gly Ala 1140 1145 1150 Thr Lys Glu Thr Leu Val Ile Asn Thr Val Thr Pro Leu Val Leu Asp 1155 1160 1165 1170 Asn Ser Lys Thr Tyr Lys Ile Val Val Ser Gly Val Lys Asp Ala Ala 1175 1180 1185 Gly Asn Val Ala Asp Thr Ile Thr Phe Tyr Ile Lys 1190 1195 3 33 DNA Artificial Sequence Description of Artificial Sequence synthetic primer 3 ttaatcgatt ctagatggat aggaaaaaag ctg 33 4 37 DNA Artificial Sequence Description of Artificial Sequence synthetic primer 4 atacccgggg gtacggatcc gatacagatt tgagcaa 37 5 2766 DNA Bacillus stearothermophilus CDS (1)..(2763) sig_peptide (1)..(93) mat_peptide (94)..(2763) 5 atg gct tat caa cct aag tct ttt cgc aag ttt gtt gcg aca act gca 48 Met Ala Tyr Gln Pro Lys Ser Phe Arg Lys Phe Val Ala Thr Thr Ala -30 -25 -20 aca gct gcc att gta gca tct gcg gta gct cct gta gta tct gca gca 96 Thr Ala Ala Ile Val Ala Ser Ala Val Ala Pro Val Val Ser Ala Ala -15 -10 -5 -1 1 agc ttc aca gat gtt gcg ccg caa tat aaa gat gcg atc gat ttc tta 144 Ser Phe Thr Asp Val Ala Pro Gln Tyr Lys Asp Ala Ile Asp Phe Leu 5 10 15 gta tca act ggt gca aca aaa ggt aaa aca gaa aca aaa ttc ggc gtt 192 Val Ser Thr Gly Ala Thr Lys Gly Lys Thr Glu Thr Lys Phe Gly Val 20 25 30 tac gat gaa atc act cgt cta gat gcg gca gtt att ctt gca aga gta 240 Tyr Asp Glu Ile Thr Arg Leu Asp Ala Ala Val Ile Leu Ala Arg Val 35 40 45 tta aaa cta gac gtt gac aac gca aaa gac gca ggc ttc aca gat gtg 288 Leu Lys Leu Asp Val Asp Asn Ala Lys Asp Ala Gly Phe Thr Asp Val 50 55 60 65 cca aaa gac cgt gca aaa tac gtc aac gcg ctt gta gaa gct ggc gta 336 Pro Lys Asp Arg Ala Lys Tyr Val Asn Ala Leu Val Glu Ala Gly Val 70 75 80 tta aac ggt aaa gca cct ggc aaa ttt ggt gca tac gac cca tta act 384 Leu Asn Gly Lys Ala Pro Gly Lys Phe Gly Ala Tyr Asp Pro Leu Thr 85 90 95 cgc gtt gaa atg gca aaa atc atc gcg aac cgt tac aaa tta aaa gct 432 Arg Val Glu Met Ala Lys Ile Ile Ala Asn Arg Tyr Lys Leu Lys Ala 100 105 110 gac gat gta aaa ctt cca ttc act gat gta aac gat aca tgg gca cca 480 Asp Asp Val Lys Leu Pro Phe Thr Asp Val Asn Asp Thr Trp Ala Pro 115 120 125 tac gta aaa gcg ctt tat aaa tac gaa gta acc aaa agg tta aaa cac 528 Tyr Val Lys Ala Leu Tyr Lys Tyr Glu Val Thr Lys Arg Leu Lys His 130 135 140 145 caa caa gct tcg gtg cat acc aaa aac atc act ctg cgt gac ttt gcg 576 Gln Gln Ala Ser Val His Thr Lys Asn Ile Thr Leu Arg Asp Phe Ala 150 155 160 caa ttt gta tat aga gcg gtg aat att aat gca gtg cca gaa ata gtt 624 Gln Phe Val Tyr Arg Ala Val Asn Ile Asn Ala Val Pro Glu Ile Val 165 170 175 gaa gta act gcg gtt aat tcg act aca gtg aaa gta aca ttc aat acg 672 Glu Val Thr Ala Val Asn Ser Thr Thr Val Lys Val Thr Phe Asn Thr 180 185 190 caa att gct gat gtt gat ttc aca aat ttt gct atc gat aac ggt tta 720 Gln Ile Ala Asp Val Asp Phe Thr Asn Phe Ala Ile Asp Asn Gly Leu 195 200 205 act gtt act aaa gca act ctt tct cgt gat aaa aaa tcc gta gag gtt 768 Thr Val Thr Lys Ala Thr Leu Ser Arg Asp Lys Lys Ser Val Glu Val 210 215 220 225 gtg gta aat aaa ccg ttt act cgt aat cag gaa tat aca att aca gcg 816 Val Val Asn Lys Pro Phe Thr Arg Asn Gln Glu Tyr Thr Ile Thr Ala 230 235 240 aca ggc att aaa aat tta aaa ggc gag acc gct aag gaa tta act ggt 864 Thr Gly Ile Lys Asn Leu Lys Gly Glu Thr Ala Lys Glu Leu Thr Gly 245 250 255 aag ttt gtt tgg tct gtt caa gat gcg gta act gtt gca cta aat aat 912 Lys Phe Val Trp Ser Val Gln Asp Ala Val Thr Val Ala Leu Asn Asn 260 265 270 agt tcg ctt aaa gtt gga gag gaa tct ggt tta act gta aaa gat cag 960 Ser Ser Leu Lys Val Gly Glu Glu Ser Gly Leu Thr Val Lys Asp Gln 275 280 285 gat ggc aaa gat gtt gta ggt gct aaa gta gaa ctt act tct tct aat 1008 Asp Gly Lys Asp Val Val Gly Ala Lys Val Glu Leu Thr Ser Ser Asn 290 295 300 305 act aat att gtt gta gtt tca agt ggc gaa gta tca gta tct gct gct 1056 Thr Asn Ile Val Val Val Ser Ser Gly Glu Val Ser Val Ser Ala Ala 310 315 320 aaa gtt aca gct gta aaa ccg gga aca gct gat gtt act gca aaa gtt 1104 Lys Val Thr Ala Val Lys Pro Gly Thr Ala Asp Val Thr Ala Lys Val 325 330 335 aca tta cca gat ggt gtt gta cta aca aat aca ttt aaa gtg aca gtt 1152 Thr Leu Pro Asp Gly Val Val Leu Thr Asn Thr Phe Lys Val Thr Val 340 345 350 aca gaa gtg cct gtt caa gtc caa aat caa gga ttt act tta gtt gat 1200 Thr Glu Val Pro Val Gln Val Gln Asn Gln Gly Phe Thr Leu Val Asp 355 360 365 aat ctt tct aat gct cca cag aat aca gtt gca ttt aac aaa gct gag 1248 Asn Leu Ser Asn Ala Pro Gln Asn Thr Val Ala Phe Asn Lys Ala Glu 370 375 380 385 aaa gta act tca atg ttt gct gga gaa act aaa aca gtt gca atg tat 1296 Lys Val Thr Ser Met Phe Ala Gly Glu Thr Lys Thr Val Ala Met Tyr 390 395 400 gat act aaa aac ggt gat cct gaa act aaa cct gtt gat ttc aaa gat 1344 Asp Thr Lys Asn Gly Asp Pro Glu Thr Lys Pro Val Asp Phe Lys Asp 405 410 415 gca act gta cgt tca tta aat cca att att gca aca gct gct att aat 1392 Ala Thr Val Arg Ser Leu Asn Pro Ile Ile Ala Thr Ala Ala Ile Asn 420 425 430 ggt agt gag ctc ctt gtc aca gct aat gct ggc caa tct gga aaa gct 1440 Gly Ser Glu Leu Leu Val Thr Ala Asn Ala Gly Gln Ser Gly Lys Ala 435 440 445 tca ttt gaa gta aca tta aaa gat aat aca aaa aga aca ttt aca gtt 1488 Ser Phe Glu Val Thr Leu Lys Asp Asn Thr Lys Arg Thr Phe Thr Val 450 455 460 465 gat gta aaa aaa gac cct gta tta caa gat ata aaa gta gat gca act 1536 Asp Val Lys Lys Asp Pro Val Leu Gln Asp Ile Lys Val Asp Ala Thr 470 475 480 tct gtt aaa ctt tcc gat gaa gct gtt ggc ggc ggg gaa gtt gaa gga 1584 Ser Val Lys Leu Ser Asp Glu Ala Val Gly Gly Gly Glu Val Glu Gly 485 490 495 gtt aac caa aaa acg att aaa gta agt gca gtt gac caa tac ggt aaa 1632 Val Asn Gln Lys Thr Ile Lys Val Ser Ala Val Asp Gln Tyr Gly Lys 500 505 510 gaa att aaa ttt ggt aca aaa ggt aaa gtt act gtt aca act aat aca 1680 Glu Ile Lys Phe Gly Thr Lys Gly Lys Val Thr Val Thr Thr Asn Thr 515 520 525 gaa gga cta gtt att aaa aat gta aat agc gat aat aca att gac ttt 1728 Glu Gly Leu Val Ile Lys Asn Val Asn Ser Asp Asn Thr Ile Asp Phe 530 535 540 545 gat agc ggc aat agt gca act gac caa ttt gtt gtc gtt gca aca aaa 1776 Asp Ser Gly Asn Ser Ala Thr Asp Gln Phe Val Val Val Ala Thr Lys 550 555 560 gac aaa att gtc aat ggt aaa gta gaa gtt aaa tat ttc aaa aat gct 1824 Asp Lys Ile Val Asn Gly Lys Val Glu Val Lys Tyr Phe Lys Asn Ala 565 570 575 agt gac aca aca cca act tca act aaa aca att act gtt aat gta gta 1872 Ser Asp Thr Thr Pro Thr Ser Thr Lys Thr Ile Thr Val Asn Val Val 580 585 590 aat gta aaa gct gac gct aca cca gta gga tta gat att gta gca cct 1920 Asn Val Lys Ala Asp Ala Thr Pro Val Gly Leu Asp Ile Val Ala Pro 595 600 605 tct aaa att gat gta aat gct cca aac act gct tct act gca gat gtt 1968 Ser Lys Ile Asp Val Asn Ala Pro Asn Thr Ala Ser Thr Ala Asp Val 610 615 620 625 gat ttt ata aat ttc gaa agt gtt gag att tac aca ctc gat tca aat 2016 Asp Phe Ile Asn Phe Glu Ser Val Glu Ile Tyr Thr Leu Asp Ser Asn 630 635 640 ggt aga cgt caa aaa aaa gtt act cca act gca act aca ctt gta ggt 2064 Gly Arg Arg Gln Lys Lys Val Thr Pro Thr Ala Thr Thr Leu Val Gly 645 650 655 aca aaa aaa aaa aaa aaa gtt aat ggg aat gta tta caa ttc aag ggg 2112 Thr Lys Lys Lys Lys Lys Val Asn Gly Asn Val Leu Gln Phe Lys Gly 660 665 670 aac gaa gaa tta acg cta tca act tct tct agt aca gga aac gta gat 2160 Asn Glu Glu Leu Thr Leu Ser Thr Ser Ser Ser Thr Gly Asn Val Asp 675 680 685 gga aca gca gaa gga atg aca aaa cgt att cca ggg aaa tat atc aac 2208 Gly Thr Ala Glu Gly Met Thr Lys Arg Ile Pro Gly Lys Tyr Ile Asn 690 695 700 705 tct gca agt gta cct gcc agt gca aca gta gca aca agt cct gtt act 2256 Ser Ala Ser Val Pro Ala Ser Ala Thr Val Ala Thr Ser Pro Val Thr 710 715 720 gta aag ctt aat tca agt gat aat gat tta aca ttt gaa gaa tta ata 2304 Val Lys Leu Asn Ser Ser Asp Asn Asp Leu Thr Phe Glu Glu Leu Ile 725 730 735 ttc ggt gta att gac cct aca caa tta gtc aaa gat gaa gac atc aac 2352 Phe Gly Val Ile Asp Pro Thr Gln Leu Val Lys Asp Glu Asp Ile Asn 740 745 750 gaa ttt att gca gtt tca aaa gcg gct aaa aat gat gga tat ttg tat 2400 Glu Phe Ile Ala Val Ser Lys Ala Ala Lys Asn Asp Gly Tyr Leu Tyr 755 760 765 aat aaa ccg ctt gta acg gtt aaa gat gca tca gga aaa gtt att cca 2448 Asn Lys Pro Leu Val Thr Val Lys Asp Ala Ser Gly Lys Val Ile Pro 770 775 780 785 aca ggt gca aat gtt tac ggt cta aat cat gat gca act aac gga aac 2496 Thr Gly Ala Asn Val Tyr Gly Leu Asn His Asp Ala Thr Asn Gly Asn 790 795 800 att tgg ttt gat gag gaa caa gct ggc tta gct aaa aaa ttt agt gat 2544 Ile Trp Phe Asp Glu Glu Gln Ala Gly Leu Ala Lys Lys Phe Ser Asp 805 810 815 gta cat ttt gat gtt gat ttt tca tta act aac gtt gta aaa act ggt 2592 Val His Phe Asp Val Asp Phe Ser Leu Thr Asn Val Val Lys Thr Gly 820 825 830 agc ggt aca gtt tct tca tcg cca tca tta tct gac gca att caa ctt 2640 Ser Gly Thr Val Ser Ser Ser Pro Ser Leu Ser Asp Ala Ile Gln Leu 835 840 845 act aat tca ggc gat gca gta tcg ttt aca tta gtt atc aaa tca att 2688 Thr Asn Ser Gly Asp Ala Val Ser Phe Thr Leu Val Ile Lys Ser Ile 850 855 860 865 tat gtt aaa ggc gca gat aaa gat gat aat aac tta ctt gca gcc cct 2736 Tyr Val Lys Gly Ala Asp Lys Asp Asp Asn Asn Leu Leu Ala Ala Pro 870 875 880 gtt tct gtc aat gtg act gtg aca aaa taa 2766 Val Ser Val Asn Val Thr Val Thr Lys 885 890 6 921 PRT Bacillus stearothermophilus 6 Met Ala Tyr Gln Pro Lys Ser Phe Arg Lys Phe Val Ala Thr Thr Ala -30 -25 -20 Thr Ala Ala Ile Val Ala Ser Ala Val Ala Pro Val Val Ser Ala Ala -15 -10 -5 -1 1 Ser Phe Thr Asp Val Ala Pro Gln Tyr Lys Asp Ala Ile Asp Phe Leu 5 10 15 Val Ser Thr Gly Ala Thr Lys Gly Lys Thr Glu Thr Lys Phe Gly Val 20 25 30 Tyr Asp Glu Ile Thr Arg Leu Asp Ala Ala Val Ile Leu Ala Arg Val 35 40 45 Leu Lys Leu Asp Val Asp Asn Ala Lys Asp Ala Gly Phe Thr Asp Val 50 55 60 65 Pro Lys Asp Arg Ala Lys Tyr Val Asn Ala Leu Val Glu Ala Gly Val 70 75

80 Leu Asn Gly Lys Ala Pro Gly Lys Phe Gly Ala Tyr Asp Pro Leu Thr 85 90 95 Arg Val Glu Met Ala Lys Ile Ile Ala Asn Arg Tyr Lys Leu Lys Ala 100 105 110 Asp Asp Val Lys Leu Pro Phe Thr Asp Val Asn Asp Thr Trp Ala Pro 115 120 125 Tyr Val Lys Ala Leu Tyr Lys Tyr Glu Val Thr Lys Arg Leu Lys His 130 135 140 145 Gln Gln Ala Ser Val His Thr Lys Asn Ile Thr Leu Arg Asp Phe Ala 150 155 160 Gln Phe Val Tyr Arg Ala Val Asn Ile Asn Ala Val Pro Glu Ile Val 165 170 175 Glu Val Thr Ala Val Asn Ser Thr Thr Val Lys Val Thr Phe Asn Thr 180 185 190 Gln Ile Ala Asp Val Asp Phe Thr Asn Phe Ala Ile Asp Asn Gly Leu 195 200 205 Thr Val Thr Lys Ala Thr Leu Ser Arg Asp Lys Lys Ser Val Glu Val 210 215 220 225 Val Val Asn Lys Pro Phe Thr Arg Asn Gln Glu Tyr Thr Ile Thr Ala 230 235 240 Thr Gly Ile Lys Asn Leu Lys Gly Glu Thr Ala Lys Glu Leu Thr Gly 245 250 255 Lys Phe Val Trp Ser Val Gln Asp Ala Val Thr Val Ala Leu Asn Asn 260 265 270 Ser Ser Leu Lys Val Gly Glu Glu Ser Gly Leu Thr Val Lys Asp Gln 275 280 285 Asp Gly Lys Asp Val Val Gly Ala Lys Val Glu Leu Thr Ser Ser Asn 290 295 300 305 Thr Asn Ile Val Val Val Ser Ser Gly Glu Val Ser Val Ser Ala Ala 310 315 320 Lys Val Thr Ala Val Lys Pro Gly Thr Ala Asp Val Thr Ala Lys Val 325 330 335 Thr Leu Pro Asp Gly Val Val Leu Thr Asn Thr Phe Lys Val Thr Val 340 345 350 Thr Glu Val Pro Val Gln Val Gln Asn Gln Gly Phe Thr Leu Val Asp 355 360 365 Asn Leu Ser Asn Ala Pro Gln Asn Thr Val Ala Phe Asn Lys Ala Glu 370 375 380 385 Lys Val Thr Ser Met Phe Ala Gly Glu Thr Lys Thr Val Ala Met Tyr 390 395 400 Asp Thr Lys Asn Gly Asp Pro Glu Thr Lys Pro Val Asp Phe Lys Asp 405 410 415 Ala Thr Val Arg Ser Leu Asn Pro Ile Ile Ala Thr Ala Ala Ile Asn 420 425 430 Gly Ser Glu Leu Leu Val Thr Ala Asn Ala Gly Gln Ser Gly Lys Ala 435 440 445 Ser Phe Glu Val Thr Leu Lys Asp Asn Thr Lys Arg Thr Phe Thr Val 450 455 460 465 Asp Val Lys Lys Asp Pro Val Leu Gln Asp Ile Lys Val Asp Ala Thr 470 475 480 Ser Val Lys Leu Ser Asp Glu Ala Val Gly Gly Gly Glu Val Glu Gly 485 490 495 Val Asn Gln Lys Thr Ile Lys Val Ser Ala Val Asp Gln Tyr Gly Lys 500 505 510 Glu Ile Lys Phe Gly Thr Lys Gly Lys Val Thr Val Thr Thr Asn Thr 515 520 525 Glu Gly Leu Val Ile Lys Asn Val Asn Ser Asp Asn Thr Ile Asp Phe 530 535 540 545 Asp Ser Gly Asn Ser Ala Thr Asp Gln Phe Val Val Val Ala Thr Lys 550 555 560 Asp Lys Ile Val Asn Gly Lys Val Glu Val Lys Tyr Phe Lys Asn Ala 565 570 575 Ser Asp Thr Thr Pro Thr Ser Thr Lys Thr Ile Thr Val Asn Val Val 580 585 590 Asn Val Lys Ala Asp Ala Thr Pro Val Gly Leu Asp Ile Val Ala Pro 595 600 605 Ser Lys Ile Asp Val Asn Ala Pro Asn Thr Ala Ser Thr Ala Asp Val 610 615 620 625 Asp Phe Ile Asn Phe Glu Ser Val Glu Ile Tyr Thr Leu Asp Ser Asn 630 635 640 Gly Arg Arg Gln Lys Lys Val Thr Pro Thr Ala Thr Thr Leu Val Gly 645 650 655 Thr Lys Lys Lys Lys Lys Val Asn Gly Asn Val Leu Gln Phe Lys Gly 660 665 670 Asn Glu Glu Leu Thr Leu Ser Thr Ser Ser Ser Thr Gly Asn Val Asp 675 680 685 Gly Thr Ala Glu Gly Met Thr Lys Arg Ile Pro Gly Lys Tyr Ile Asn 690 695 700 705 Ser Ala Ser Val Pro Ala Ser Ala Thr Val Ala Thr Ser Pro Val Thr 710 715 720 Val Lys Leu Asn Ser Ser Asp Asn Asp Leu Thr Phe Glu Glu Leu Ile 725 730 735 Phe Gly Val Ile Asp Pro Thr Gln Leu Val Lys Asp Glu Asp Ile Asn 740 745 750 Glu Phe Ile Ala Val Ser Lys Ala Ala Lys Asn Asp Gly Tyr Leu Tyr 755 760 765 Asn Lys Pro Leu Val Thr Val Lys Asp Ala Ser Gly Lys Val Ile Pro 770 775 780 785 Thr Gly Ala Asn Val Tyr Gly Leu Asn His Asp Ala Thr Asn Gly Asn 790 795 800 Ile Trp Phe Asp Glu Glu Gln Ala Gly Leu Ala Lys Lys Phe Ser Asp 805 810 815 Val His Phe Asp Val Asp Phe Ser Leu Thr Asn Val Val Lys Thr Gly 820 825 830 Ser Gly Thr Val Ser Ser Ser Pro Ser Leu Ser Asp Ala Ile Gln Leu 835 840 845 Thr Asn Ser Gly Asp Ala Val Ser Phe Thr Leu Val Ile Lys Ser Ile 850 855 860 865 Tyr Val Lys Gly Ala Asp Lys Asp Asp Asn Asn Leu Leu Ala Ala Pro 870 875 880 Val Ser Val Asn Val Thr Val Thr Lys 885 890 7 498 DNA Unknown Organism Description of Unknown Organism streptavidin gene 7 cccatggacc cgtccaagga ctccaaagct caggtttctg cagccgaagc tggtatcact 60 ggcacctggt ataaccaact ggggtcgact ttcattgtga ccgctggtgc ggacggagct 120 ctgactggca cctacgaatc tgcggttggt aacgcagaat cccgctacgt actgactggc 180 cgttatgact ctgcacctgc caccgatggc tctggtaccg ctctgggctg gactgtggct 240 tggaaaaaca actatcgtaa tgcgcacagc gccactacgt ggtctggcca atacgttggc 300 ggtgctgagg ctcgtatcaa cactcagtgg ctgttaacat ccggcactac cgaagcgaat 360 gcatggaaat cgacactagt aggtcatgac acctttacca aagttaagcc ttctgctgct 420 agcattgatg ctgccaagaa agcaggcgta aacaacggta accctctaga cgctgttcag 480 caataataag gatccggg 498 8 29 DNA Artificial Sequence Description of Artificial Sequence synthetic primer 8 ttcatcgtaa acgccgaatt ttgtttctg 29 9 26 DNA Artificial Sequence Description of Artificial Sequence synthetic primer 9 agggaaatat atcaactctg caagtg 26 10 49 DNA Bacillus stearothermophilus 10 gaattcatcg atgtcgacca aggaggtcta gatggatccg gccaagctt 49

Claims

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.