DE19910102B4 - Protein conjugates, methods, vectors, proteins and DNA for their production, their use, and medicines and vaccines containing the same - Google Patents

Abstract

Protein conjugate consisting of at least one functional area at any one Position of the sequence of a carrier protein region for forming a capsid space structure of the lumazine synthase type, so that their outer periphery is covalently linked to a plurality of these functional areas.

Description

The The invention relates to protein conjugates, methods, vectors, proteins and DNA for their production, as well as their use, as well as pharmaceuticals or vaccines containing them. The present invention is used for the preparation of supramolecular particles, which one or several different, arbitrarily determinable Structural units in large Number on the surface a single, approximately spherical protein molecule present.

Properties of lumazine synthase and lumazine synthase based artifact protein conjugates

WHERE 99/38986 describes a plant lumazine synthase gene and a probable amino acid sequence lumazine synthase from A. haliava.

6,7-Dimethyl-8-ribityllumazine synthase (hereinafter referred to as lumazine synthase) catalyzes the penultimate step of vitamin B ₂ biosynthesis in microorganisms and plants.

lumazine synthases from certain bacteria (e.g., Escherichia coli, Bacillus subtilis, Aquifex aeolicus) form highly symmetric, icosahedral complexes out of 60 subunits with a molecular weight of about 1 MDalton (Bacher and Ladenstein, 1991; Bacher et al., 1980; Ladenstein et al., 1986, 1988, 1994; Mörtl et al., 1996). X-ray structures the capsid shell lumazine synthase from Bacillus subtilis are known (Ladenstein et al., 1988, 1994; Ritsert et al., 1995). The protein from Bacillus subtilis can be denatured using urea and then renatured become. The efficiency of renaturation can be improved by adding a Ligands (substrate analog), for example 5-nitro-6- (D-ribitylamino) -2,4- (1H, 3H) -pyrimidinedione or 5-nitroso-6- (D-ribitylamino) -2,4- (1H, 3H) -pyrimidinedione. The Folding the renatured protein is identical to the folding the native lumazine synthase. In the presence of said ligand the lumazine synthase from Bacillus subtilis is stable up to pH 10. The environment of the inhibitor molecule is due to the X-ray structure known. The binding site of this ligand becomes more adjacent to segments Monomers (Bacher et al., 1986; Ritsert et al., 1995). This constellation explains the supporting one Influence of the ligand during the renaturation of the high molecular weight protein complex.

lumazine synthases from different microorganisms can be found in recombinant strains of Escherichia coli and Bacillus subtilis are efficiently expressed. The recombinant Proteins can be isolated in high yield.

Either the N- as well as the C-terminus lie on the surface of the icosahedral capsid molecule. For lumazine synthase from Bacillus subtilis, this was first confirmed by X-ray diffraction (Ladenstein et al., 1988). By DNA synthesis could a optimally adapted to the codon usage of Escherichia coli Gene for expressing the thermostable lumazine synthase from the thermophilic Microorganism Aquifex aeolicus be obtained. The protein can be obtained recombinantly in high amounts. It's at a temperature from 80 ° C stable for at least a week. That in Aquifex aeolicus the same structural conditions as is the case with lumazine synthase from Bacillus subtilis, follows from the fact that fusion proteins with extension of the C-terminal and / or N-terminal May eventually associate with icosahedral capsids, or that chimeric proteins, consisting of parts of the lumazine synthases from Aquifex aeolicus and Bacillus subtilis can be produced. It is therefore of one very similar Quaternary structure.

icosahedral Lumazine synthases can on their surface be functionalized with structural units. As structural units (Biomolecules) preferably come oligo- or Polypeptides with freely selectable segments into consideration. The presented Proteins (conjugated biomolecules) are with the carrier protein covalently linked (lumazine synthase conjugate). Under a carrier protein For the purposes of the invention, a natural (unaltered) or a modified lumazine synthase, in which the primary structure changed has been. It can one or more amino acids replaced and / or removed and / or added and / or changed. The receipt of the original catalytic activity the lumazine synthase is not required. Rather, e.g. also catalytically inactive, modified proteins for all applications according to the invention be used.

The number of conjugated biomolecules on the surface of the carrier protein can extend over a wide range, according to the invention the surface having up to 60 (at one terminus) or 120 (at both termini) or 180 (at both termini plus loop insertion) identical or different in their structure peptide motifs can be decorated. In addition, protein subunits on the structural basis of lumazine synthase can also be assembled into even larger, for example, spherical particles and tubular structures. These associates can contain well over 60 subunits. However, they do not possess the strict geometrical regularity of icosahedral 60-mer lumazine synthase molecules.

The Length of presented Peptide segments can over vary a wide range, according to the invention preferably over 1-500 amino acids, wherein the peptide motifs both unchanged as well as modified.

Proteins of the invention can also one or more amino acid analogues or not naturally occurring amino acids obtained by biological means (e.g., by suppressor tRNA techniques, etc.). or by chemical routes (e.g. Coupling reagents, etc.) are introduced into the sequence. Likewise, modifications can be made (e.g., glycosylation, etc.) or derivatization (e.g., biotinylation, etc.).

The underlying genetic information for the specification of the artificial peptide segments introduced into the structure of the lumazine synthase subunit can range from a few codons to several genes, dependent whether an oligopeptide, a polypeptide or one of several Subunits existing protein to be encoded.

The surface A lumazine synthase can also be chemically modified so that their outer periphery is covalently linked to a variety of functional areas. The Preparation of Heterooligomeric Lumazine Synthase Conjugates over a dissociation step and a subsequent folding / reassociation step. The monomerically present after denaturation chimeric proteins can be arbitrary be mixed. Since the recombined subunits each have a possess constant Lumazinsynthaseanteil is a renaturation the lumazine synthase core structure to form the natural icosahedral structure possible.

Immunological analysis method based on the ELISA test system

Bind antibodies with high specificity to certain target structures (antigens). There were test procedures designed to detect specific antibody-antigen complexes based. To determine if an antibody is bound to its target antigen There are different possibilities. The enzyme-linked immunoabsorbent (enzyme-linked immunoabsorbent assay, ELISA) is one of these methods. The ELISA can in principle for the determination of any antigen, hapten or antibody his application today he has mainly in clinical Found biochemistry. One measures so for example hematological factors and the concentrations of serum proteins, e.g. immunoglobulins, oncofetal Proteins and hormones, e.g. Insulin. In the diagnosis of infectious diseases are bacterial toxins, microorganisms like candida albicans, Rotaviruses, herpesviruses, HIV or hepatitis B surface antigen proved in this way. Furthermore, they are immunochemical Analysis method for antibody determination, in order Diagnosis of past or ongoing infectious diseases (e.g. with HIV, hepatitis).

• 1. Die Probe, die ein spezifisches Molekül oder einen bestimmten Organismus enthalten soll, wird an einen festen Träger gebunden (z.B. Mikrotiterplatte aus Kunststoff).
• 2. Der Nachweis eines Antigens (Protein, Peptid, Hapten-Konjugat, etc.) erfolgt durch die spezifische Bindung eines spezifischen Antikörpers (primärer Antikörper), der gegen das betreffende Antigen aus 1. gerichtet ist. Hierbei kann der primäre Antikörper selbst markiert sein (z.B. radioaktiv) und daher unmittelbar lokalisiert werden (z.B. Autoradiographie). Alternativ kann entsprechend dem folgenden Absatz weitergearbeitet werden.
• 3. Häufig erfolgt stattdessen die Zugabe eines zweiten Antikörpers (sekundärer Antikörper), der spezifisch an den primären Antikörper, aber nicht an das Antigen aus 1., bindet. Dieser zweite Antikörper ist häufig chemisch mit einem Enzym gekoppelt (Indikatorsystem), welches eine Umwandlung eines farblosen Substrates in ein farbiges Produkt katalysiert (z.B. Alkalische Phosphatase, Meerrettich-Peroxidase, etc.). Der zweite Antikörper ist meistens gegen den konstanten Abschnitt des ersten Antikörpers gerichtet. Nicht gebundene sekundäre Antikörper werden durch Waschen entfernt.
• 4. Zugabe eines farblosen Substrats und dessen Umwandlung in ein farbiges Produkt.

An ELISA protocol generally consists of the following substeps.

• 1. The sample intended to contain a specific molecule or organism is bound to a solid support (eg plastic microtiter plate).
• 2. The detection of an antigen (protein, peptide, hapten conjugate, etc.) is carried out by the specific binding of a specific antibody (primary antibody), which is directed against the relevant antigen from 1.. In this case, the primary antibody itself may be labeled (eg radioactive) and therefore localized directly (eg autoradiography). Alternatively, work can continue according to the following paragraph.
• 3. Frequently, instead, a second antibody (secondary antibody) is added which binds specifically to the primary antibody but not to the antigen of 1.. This second antibody is often chemically coupled with an enzyme (indicator system), which catalyzes a conversion of a colorless substrate into a colored product (eg alkaline phosphatase, horseradish peroxidase, etc.). The second antibody is mostly directed against the constant portion of the first antibody. Unbound secondary antibodies are removed by washing.
• 4. Add a colorless substrate and convert it to a colored product.

He follows no binding of the primary antibody to the antigens present in the sample, the primary antibody in the first wash step away. Thus, the enzyme-labeled secondary antibody may also be do not bind, i. the test batch remains colorless. Is that concerned antigenic structure present, so the primary antibody can bind and to these the secondary Antibody. The coupled to the second antibody Enzyme catalyzes the color reaction, the product of which is easily detected leaves (e.g. photometric, etc.). The measured amount of enzyme activity is the Content of specific antigen or antibody proportional (from: Glick, B., Pasternak, J., Molecular Biotechnology, Spectrum Academic Verlag, 1995, p. 201 ff).

at the implementation binding assays require an indicator system (e.g., horseradish peroxidase) which a visualization of the elapsed immune response allowed. The Visualization is based on a stable link between the examined Reactants (antigen or antibody) and an indicator system. As indicators (amplifiers) are used: fluorescent dyes, luminescent dyes, radioactivity, enzymes, etc. The indicators can covalently or non-covalently bound to the respective reactants available. As stable non-covalent linkages between indicator and the wanted reactant serve e.g. the antigen-antibody binding, biotin-avidin binding or lectin binding.

at direct method is primary antibody with indicator covalent connected.

The indirect arrangement bypasses the labeling of the primary antibody. Of the Primary antibody is going through an antibody, which is marked with an indicator detected. This secondary antibody, the obtained from another animal species binds to all the primary antibodies each any specificity from the first species.

A another possibility The detection is a method in which three antibodies in succession to Use come. The primary antibody off Species A is an unmarked one Secondary antibodies off Species B, which is in excess detected. Subsequently follows the addition of a tertiary antibody Species A, which is linked to an indicator. The secondary antibody (compound antibody) acts as a bridge between primary and tertiary antibodies. By the use of multiple cascaded antibodies will an increase in sensitivity.

The Visualization of the bound primary antibody may alternatively by other binding systems take place. A suitable system represents the Avidin-biotin complex binding (ABC system). Here, the primary or the secondary antibody is biotinylated available. The indicators are also biotinylated and become the tetravalent avidin saturating three binding sites bound. The fourth avidin-binding site can be biotinylated to the Primary- or bind secondary antibodies. Are the indicators used repeatedly biotinylated, revealed very big ones Avidin-enzyme complexes that increase the sensitivity of the test system (instead avidin can also be used streptavidin). (from bioanalysis, F. Lottspeich, H. Zorbas, Spektrum Akademischer Verlag, 1998, p. 91 ff) The problem with this process is a further increase the sensitivity.

• 1. Durch Zwischenschaltung eines biotinylierten multimeren Lumazinsynthase-Konjugats (Linkerprotein) zwischen Primärantikörper und Indikator: Eine Lumazinsynthase, die bis zu 60 Biotinmoleküle (z.B. über einen kurzen Abstandshalter an die Lumazinsynthase gebunden, um eine mögliche sterische Hinderung zu vermeiden) auf ihrer Oberfläche enthält, nimmt dabei aufgrund ihrer sphärischen, multimeren Struktur eine besondere Stellung ein. Die Bindung zwischen Antikörper und Linkerprotein bzw. zwischen Linkerprotein und Indikator erfolgt dabei unter Verwendung einer Avidinbrücke oder einer Streptavidinbrücke. Alternativ dazu, können auch Avidin- oder Streptavidin-markierte Primärantikörper bzw. Indikatoren zum Einsatz kommen. An 59 der 60 Biotinmoleküle auf dem multimeren Linkerprotein können Indikatormoleküle gebunden werden, wobei lediglich ein Biotinmolekül zur Vermittlung einer Bindung zwischen Primärantikörper und Linkerprotein notwendig ist. Durch die resultierende mehrfache Bindung von farbreaktionvermittelndem Enzym, wird eine extreme Signalverstärkung erreicht, wobei die Signalstärke proportional zur Antigenkonzentration ansteigt.
• 2. Durch Einsatz von heterooligomeren biotinylierten Lumazinsynthase-Konjugaten: Durch die erfindungsgemäße Reassoziation von unterschiedlichen Lumazinsynthase-Varianten (beispielsweise eine Kombination von 1–3 Antigen-enthaltenen Lumazinsynthasemonomeren mit bis zu 59 biotinylierten Lumazinsynthasemonomeren), wird ein heterooligomeres Lumazinsynthase-Konjugat erzeugt, welches sowohl einen Reaktanden (z.B. Antigen) als auch mehrere Biotin-Moleküle enthält. An die Biotin-Moleküle können Streptavidin- oder Avidin-vermittelt (oder auch über einen Anti-Biotin-Antikörper) Indikatormoleküle gebunden werden. Exemplarisch sollen im folgenden zwei Einsatzmöglichkeiten besprochen werden: A) Ein Lumazinsynthase-Konjugat, welches 1–5 kurze Peptide von antigen wirksamen viralen oder bakteriellen Oberflächenproteinen (antigene Determinanten) und bis zu 60 Biotinmoleküle kovalent gebunden enthält, dient als Detektionsmolekül für immobilisierte Antikörper, die aus einem Patientenserum oder anderen Flüssigkeiten stammen. B) Charakteristische Antikörper gegen bestimmte Infektionskrankheiten werden mit Hilfe spezieller immobilisierter Epitope (Teile von Oberflächenproteinen der betreffenden pathogenen Organismen; antigene Determinanten) aus der jeweiligen Körperflüssigkeit gefischt. Ein Lumazinsynthase-Konjugat, welches ebenfalls 1–5 Kopien des oben bezeichneten Epitops und bis zu 60 Biotinmoleküle kovalent gebunden enthält, dient als Detektionsmolekül für die, an das immobilisierte Epitop gebundenen, Antikörper. Eine Farbreaktion wird in beiden Fällen (A und B) durch ein beliebiges streptavidingekoppeltes Enzym, welches einen Komplex mit der biotinylierten Lumazinsynthase eingeht, erreicht. Durch die Zwischenschaltung dieses mehrfach biotinylierten Linkerproteins (Lumazinsynthase-Konjugat) und der dadurch bedingten mehrfachen Bindung von farbreaktionvermittelndem Enzym, wird eine Signalverstärkung erreicht.
• 3. Durch Einsatz von heterooligomeren, nicht biotinylierten, Lumazinsynthase-Konjugaten: Durch die erfindungsgemäße Reassoziation von unterschiedlichen Lumazinsynthase-Varianten wird ein heterooligomeres Lumazinsynthase-Konjugat erzeugt, welches sowohl einen Reaktanden (z.B. Antigen, welches spezifisch Antikörper aus einem Patientenserum binden kann) in einfacher Ausführung, als auch Epitope in mehrfacher Ausführung, welche von Indikator-markierten Antikörpern erkannt werden, enthält. Auch hierbei wird, durch die mögliche mehrfache Bindung von Antikörper-Indikator-Komplexen an das multimere Protein, eine Signalverstärkung erreicht.

Signal amplification through the use of a derivatized, multimeric lumazine synthase in solution or on any surface:

• 1. By interposing a biotinylated multimeric lumazine synthase conjugate (linker protein) between primary antibody and indicator: a lumazine synthase containing on its surface up to 60 biotin molecules (eg bound to the lumazine synthase via a short spacer to avoid possible steric hindrance), it occupies a special position due to its spherical, multi-faceted structure. The binding between antibody and linker protein or between linker protein and indicator takes place using an avidin bridge or a streptavidin bridge. Alternatively, avidin or streptavidin labeled primary antibodies or indicators may be used. Indicator molecules can be bound to 59 of the 60 biotin molecules on the multimeric linker protein, with only one biotin molecule being required to mediate binding between the primary antibody and the linker protein. The resulting multiple binding of color reaction-mediating enzyme achieves extreme signal enhancement, with signal strength increasing in proportion to the antigen concentration.
• 2. By using heterooligomeric biotinylated lumazine synthase conjugates: The reassociation of different lumazine synthase variants according to the invention (for example a combination of 1-3 antigen-containing lumazine synthase monomers with up to 59 biotinylated lumazine synthase monomers) produces a hetero-oligomeric lumazine synthase conjugate which is both contains a reactant (eg antigen) as well as several biotin molecules. The biotin molecules can be strepta vidin- or avidin-mediated (or via an anti-biotin antibody) indicator molecules are bound. By way of example, two possible uses are to be discussed below: A) A lumazine synthase conjugate containing 1-5 short peptides of antigenically active viral or bacterial surface proteins (antigenic determinants) and up to 60 biotin molecules covalently bound serves as immobilized antibody detection molecule come from a patient's serum or other fluids. B) Characteristic antibodies against certain infectious diseases are fished out of the respective body fluid with the help of special immobilized epitopes (parts of surface proteins of the respective pathogenic organisms, antigenic determinants). A lumazine synthase conjugate, which also contains 1-5 copies of the above-identified epitope and up to 60 biotin molecules covalently bound, serves as a detection molecule for the antibody bound to the immobilized epitope. A color reaction is achieved in both cases (A and B) by any streptavidin-coupled enzyme which complexes with the biotinylated lumazine synthase. Through the interposition of this multiply biotinylated linker protein (lumazine synthase conjugate) and the resulting multiple binding of color reaction-mediating enzyme, signal amplification is achieved.
• 3. The use of heterooligomeric, non-biotinylated, lumazine synthase conjugates: The reassociation according to the invention of different variants of lumazine synthase produces a hetero-oligomeric lumazine synthase conjugate which can simplify both a reactant (eg antigen which can specifically bind antibodies from a patient's serum) Execution, as well as epitopes in multiple execution, which are recognized by indicator-labeled antibodies contains. Again, signal amplification is achieved by the possible multiple binding of antibody-indicator complexes to the multimeric protein.

biosensors

Classical biochemical analysis methods, e.g. the immunoassay, based on chemical reaction systems in the liquid state. A possible alternative offers the use of solid phase measuring devices or biosensors. In recent years, the use of biosensors as fast and sensitive test systems for the detection of various Substance and molecular classes more and more application. A biosensor consists of at least three Ingredients: biological receptor, transducer and associated electronics. In an immune sensor, the biological receptor may be an antibody or an antigen that is coupled to the transducer in several ways is. In both mentioned variations the sensors allow the measurement specifically forming antigen-antibody complexes.

For use as chemical sensors in liquids, such as serums, volumetric oscillators are particularly suitable. These include the quartz crystals, which are coated on a specially treated surface (depending on the test principle) with antigenic proteins or monoclonal antibodies. If an electrical alternating voltage is applied to these quartz crystals, the crystal is excited into elastic oscillations whose amplitude reaches a maximum when the electrical frequency coincides with one of the mechanical natural frequencies of the respective quartz. These vibrations can be detected with suitable measuring systems. If an antigen-coated quartz crystal is placed in a solution containing specific binding antibodies, they will attach to its surface and alter its mass. This achieves a change in its oscillation frequency and thus indicates the binding of an antibody. In addition to these piezoelectric immune sensors trying to develop measuring techniques whose operation is similar to those of potentiometric electrodes, which are therefore similar to pH meters. In this case, one aims to determine the change in potential arising from the formation of the antigen-antibody complexes on a thin equilibrated silica gel layer on the surface of the pH glass membrane. Another possibility of immunosensory measurement consists in the immobilization of proteins (antibodies or antigens) on the surface of a fiber opti's. The most commonly used optical phenomena in this method are interfering waves and surface plasmons. An interfering wave is formed when light traveling along an optical fiber is internally reflected. This interfering wave is the electromagnetic energy that arises at the interface of fiber optics and fluid. The energy is absorbed when absorbing molecules occur at the interface so that the degree of absorption is proportional to the amount of absorbent material of the interface. The formation of antigen-antibody complexes wherein the antigen or antibody is bound to the fiber surface can thus be detected. In 'Surface plasmon resonance', a metal-coated glass is used as an optical device in which an internally fully reflected light beam causes an induced electromagnetic surface wave or plasmon. Detectable surface plasmon resonance occurs at a certain angle of incidence of the light that is critically dependent on the refractive index of the medium adjacent to the metal film. Thus, changes in this layer, such as might be expected after the formation of antigen-antibody complexes, can be measured the.

To The potentiometric immune sensors are the ion-sensitive field-effect transistors to count. A Receptor (antibody, Antigen or other receptor) is doing on the semiconductor gate anchored to the transistor. The binding of an analyte to the receptor layer gets a change in the charge distribution and thus a circuit of the field effect transistor out. (From Modrow S., Falke D., Molecular Virology, Spectrum Academic Publishing House, Heidelberg, Berlin, Oxford, p. 108; Liddell E., Weeks I., Antibody Techniques, Spectrum Academic Publishing, Heidelberg, Berlin, Oxford, p. 154 ff). Also In these sensor methods, there is a desire to increase the Sensitivity.

Signal amplification through the use of derivatized, multimeric lumazine synthase molecules on a signal-transmitting surface:
Artificial lumazine synthase-based protein molecules can serve as a carrier protein in the construction of a biosensor, for example for the presentation of antigenically active capture peptides, for the detection of antibodies to certain infections. The inventive formation of mixed lumazine synthase conjugates allows the respective peptides to be incorporated into an icosahedral structure together with a binding-mediating biotin molecule. In this way, up to 59 identical or different antigenic peptides (eg domains of viral surface proteins) in association with a biotin molecule can be presented on an icosahedral molecule. By using several different multimeric lumazine synthase conjugates, a representative peptide library can be placed on a single sensor. The binding of the multimeric lumazine synthase conjugates to the surface of a transducer can be made possible for example via a streptavidin / biotin coupling.

The Sensitivity of such a test system is by offering significantly increased by several antigenic determinants, as a result not just one, but several educated against a particular pathogen antibody be recorded. Furthermore, there is no detailed knowledge about protein sections necessary, which contribute to the binding of antibodies, since with limited Expense multiple proteins of the relevant pathogen on the sensor presents can be. Since the streptavidin / biotin coupling for all epitope presentations can be used to build a biosensor always the same, namely avidin or streptavidin-coated surfaces, i. the instrumental Equipment does not have to changed become. The individual, epitope-presenting or biotinylated Lumazine synthase subunits can be easily produced by recombinant means. This has essential Benefits of developing or evaluating such detection methods.

By the presence of up to 59 capture peptides in a molecule can the surface a sensor chip (e.g., field effect transistor, surface plasmon transducer surface, etc.) be extremely enlarged and thereby an enormous increase in sensitivity can be achieved. stability problems and unspecificities are in the use of a thermostable carrier protein and the biotin / streptavidin system not to be expected.

As well can also small molecules be bound to the surface by simple chemical coupling. As coupling places stand for this singular, exposed reactive amino acids on the surface of the spherical Protein available.

Basic structure of a layer system based on a multimeric lumazine synthase:
A functionalized lumazine synthase is linked to 60 identical or differently modified subunits via an anchor (peptide, fatty acid, etc.) with a surface (for example transducer surface or any other surface that is already on a transducer). The detection sensitivity for foreign molecule binding on the surface of the lumazine synthase is thereby increased by a high number of functional groups (for example epitopes for antibody recognition, antibodies for the detection of foreign molecules in solution or other receptors).

Production of vaccines (in vitro)

vaccinations to lead to an immunological resistance to pathogens. vaccines serve mostly for prevention, this means, they should build up protection for immunized persons, they contact the respective pathogen before infection and thus protects against the disease. The injected or orally administered vaccine performs in the organism for the formation of antibodies and / or to a cellular immune response. As a result, at a future Exposure to the infectious Killed organism or neutralized, so that the Illness does not break out.

infections with bacteria, viruses, fungi and protozoa are a major factor worldwide the morbidity and mortality. Due to the increasing development of resistance against just about everybody available Antibiotics are also exacerbated in industrialized countries morbidity situation expected. The development of novel vaccines is also why of the greatest medical Importance.

When Vaccines include attenuated viruses. Attenuated viruses are similar the disease-causing agents, they are different from them however, in terms of virulence behavior; they only cause a limited or weakened one Infection and thereby induce the formation of neutralizing antibodies and cytotoxic T cells. The molecular basis of attenuation are mutations in the genome of wild-type viruses. Confer attenuated viruses usually a very good vaccine protection, obtained over several years However, they carry the risk that they in the course of the weakened infection to Revert back to wild type form can.

A another possibility for immunization in humans and animals is in the presentation antigenic parts of viral surface proteins on others, non-pathogenic viruses, e.g. Plant viruses. The gene fragments, the for an antigenic determinant (e.g., surface protein) of the pathogenically-acting Virus coding will be in the genome of non-pathogenic Virus incorporated (Dalsgaard et al., 1997). The foreign protein will together with a viral protein on the surface of the non-pathogenic virus presents. It can, however not arbitrarily large DNA fragments are integrated into the viral genome. Therefore you have to exactly Know which proteins of the virus against which infection is a vaccine should be generated for the trigger a protective one Immune response are important. Such a vaccine can not be the Generate the breadth of an immune response at the end of an infection arises with the wild-type virus or its attenuated variant. In this type of recombinant vaccine viruses is limited immunological response to a selected protein.

vaccines consisting of synthetic peptides with a length of 15 to 30 amino acids, represent a vaccine form that is currently being tested. Here are single epitopes of viral proteins affecting the formation of neutralizing antibodies effect, selected and chemically synthesized. Condition is also in this case a detailed knowledge about the protein sections that have a virus-neutralizing immune response can cause. Due to the high genetic variability of most viruses and the different ability individuals to recognize certain protein regions immunologically, would have to in a synthetic peptide-based vaccine several different Epitopes are combined with each other. As it is apart from aluminum hydroxide not a suitable human-grade adjuvant that regulates the immune response strengthen sufficiently There is still no vaccine available on synthetic Peptides: (From Modrow S., Falke D., Molecular Virology, Spektrum Akademischer Verlag, Heidelberg, Berlin, Oxford, p. 87 ff)

in the Unlike short peptides, high molecular weight molecules are suitable, i.a. Proteins and carrier-fixed Peptides, as they are administered without the use of excipients can be but still a good immunity deliver, exquisite as vaccines. The redundant presence of antigenic determinants in high numbers, as e.g. to observe in viruses or bacteria are on the immunogenic high molecular weight molecule favors the desired high antigenicity i.e. the preventive Immune response. Suitable as carrier protein for this, especially because of their icosahedral structure, the lumazine synthase. The lumazine synthase consists of at least 60 subunits, i. at least 60 identical or different antigenic determinants can be in a molecule present. The lumazine synthase is of high molecular weight and has one with some virus comparable surface structure, i. a high antigenicity is to be expected. Such vaccines are free of viral genes and they can be with relatively little effort in high quantities produce. Because big Portions of viral proteins are presented can be is in this case no well-founded detailed knowledge of the protein sections, which may cause a virus-neutralizing immune response required.

The genetically engineered proteins which are the subject of the present invention are based on the covalent linking of a wild-type lumazine synthase sequence or a modified lumazine synthase with partial structures of viruses, bacteria, fungi, protozoa or toxins. The linkage can take place at the N-terminus and / or at the C-terminus of the lumazine synthase. In addition, the peptides to be presented may be inserted into the sequence at appropriate locations on the lumazine synthase subunit so that they are presented in the form of a loop on the surface of the multi-subunit protein. Thus, a given immunological determinant can be presented in a defined high number, eg 60 times or 120 times, according to the invention, on an icosahedral molecule of 60 subunits with the triangulation number T = 1. In addition, higher molecular associates can be made who those which contain more than 100 subunits (triangulation number T = 2 or higher) and thus can present an even larger number of epitopes.

The inventive association subunits with different, genetically grafted Peptide or protein sequences also provide the opportunity to produce Protein molecules which several different antigenic sequences on a given molecule present can.

DNA vaccine

since The beginning of the 1990s will also see the possibility of using DNA examined as a vaccine. The used nucleic acids contain Genes or parts of genes of a pathogenic organism that is an immunogenic Specify protein. For The development of this vaccine is detailed knowledge of the immunological important components of great Use. The genes used predominantly encode the surface components of a pathogen or even for Parts of bacterial toxins. They will be together with regulatory Elements for controlling their expression integrated into a vector system and injected intramuscularly as purified DNA and expressed there. In particular, in muscle cells, the DNA over long periods as Episome detectable, as they obviously mined only very slowly becomes. When the appropriate genes are expressed, the organism can develop both a humoral and a cellular immune response. This form of vaccine has so far been tested in the animal system.

gene constructs, which fusion proteins consisting of protein components pathogenic Microorganisms and lumazine synthase are basically described as DNA vaccine suitable. A DNA vaccine consisting of a gene coding for a lumazine synthase (particle-forming component) and a selected gene of the pathogen can be intracellular can be expressed and thus a relatively long-lasting production of the immune system stimulating antigen. After the previous one Experiences with lumazine synthase from different organisms should the assembly of the icosahedron in vivo without auxiliary molecules (cf. Chaperonins) possible be.

Oral vaccines on plant Base

is known against which immunologically important protein component of the Pathogen a protective immune response that can be induced for this peptide encoding gene into a eukaryotic expression vector be introduced. After transformation of plant cells with this DNA can transgenic plants are expressed which express the gene in question. By eating parts of this transgenic plant is also the selected Protein component in the body and can induce an immune response there.

When particulate-forming protein is particularly suitable lumazine synthase, to the parts of immunologically active proteins of the pathogen are merged. By using a thermostable, particle-forming Lumazine synthase (e.g., from Aquifex aeolicus) as a carrier protein even a high-boiling vaccine can be produced.

Multifunctionally derivatized Immunotherapeutics based on multimeric lumazine synthase

vaccines should increase the multiplication of a pathogen and thus an infection prevent. In some cases It is difficult to be reliable Vaccine to develop because the pathogenic organism for antibodies not accessible is, or as in the case of acquired immunodeficiency syndrome (AIDS) too little about the pathogen (HIV) is known. The target cells of HIV are helper T lymphocytes (Helper cells) of the immune system, with the main functions disrupted these cells become. When HIV invades a helper cell, the virus is present protected against immunological attack. In the further course of the Disease can affect the infected cell through the production and the Release of HIV particles are destroyed. An infected one Cell can become a "factory" for production be further virus particles. The consequence of an HIV infection Above all, that is Immune system no longer protects against common infectious diseases can offer. The first step in HIV infection is the interaction a 120 kDalton glycoprotein (gp 120) from the virus envelope and the CD4 receptor on the surface the helper cells.

Antibodies to CD4 block in vitro HIV infection of helper cells. Even with an excess of free CD4 protein, the infection rate goes back in vitro. The development of a fusion protein from part of the CD4 protein and from the Fc portion of an immunoglobulin is an attempt to achieve both the protection of the helper cells and the killing of the virus. The fusion protein becomes CD4-immunoad called häsin. The molecule binds to gp120 and blocks the HIV; Both are functions of the CD4 portion. However, the ability of the fusion protein to bind to cells with Fc receptors and the long plasma half-life are due to the immunoglobulin moiety. Upon binding of the immunoadhesin to the free virus or to HIV-infected cells, an antibody-dependent, cell-mediated, cytotoxic reaction will result which will destroy the virus or the HIV-infected cell. (from: Glick, B., Pasternak, J., Molecular Biotechnology, Spektrum Akademischer Verlag, 1995, p. 245).

By the use of a multimeric derivatized lumazine synthase could improve the efficiency of this strategy. It can be one functionalized lumazine synthase which have both CD4 protein levels, as well over Fc shares. As per molecule not just a functional unit, but many of these units, should be able to increase efficiency significantly.

Instead of of the CD4 share could Also, antibodies (e.g., specially designed "single-chain antibodies") to the multimeric protein, e.g. specifically against a tumor marker (e.g., against the teratocarcinoma antigen) are directed to introduce and use this functionalized fusion protein for cancer therapy.

A further application possibility is the combination of an antibody against a tumor marker with metallothionein. The multimeric lumazine synthase is doing an anti-tumor antibody and up to 59 metallothionein molecules decorated. The metallothionein molecules in turn become radioactive Elements (with a relatively short half-life) necessary for radiotherapy are suitable (for example Technetium 99) loaded. During the Therapy binds the protein complex via its antibody component to the tumor and leads while the radiation source zoom directly to the tumor tissue. Similar Constructs can also for diagnostic purposes, e.g. for the radioactive detection of a possible Cancer disease, are used.

Use of lumazine synthase conjugates in the characterization and purification of antibodies

The The basis of foreign peptides form DNA sequences that are specific to a particular Epitope (antigenic determinant) code. The sequence of additional Peptide segments can be exact by appropriate choice of the DNA sequence be determined. It can but also peptide sequences are incorporated, which in their entire Length or have in sections a stochastic amino acid sequence. A diverse, chance-oriented variability these peptides can be modified by the use of synthetic oligonucleotides, the one about fortuitously (randomized) sequence sequence can be achieved, so that representative peptide libraries arise. This random generated peptides according to the invention on the surface of Lumazine synthase presented and are therefore synonymous for an antibody binding accessible.

The obtained Lumazinsynthase variants (with a randomized variability the foreign peptide) e.g. used to characterize antibody binding sites become. By isolating antigen-antibody complexes followed by sequencing of the bound peptide portion (N-terminal Edman sequencing or Sequence determination by mass spectroscopy), the selectivity of the binding site an antibody be characterized.

Furthermore can target antibodies, the above a specific antigen recognition (antigen sequence known in this case) feature, be searched. By using mixed conjugates, i. Lumzinsynthase conjugates both over a desired one Foreign peptide (in multiple version), as well over have a biotinylated portion (in a simple embodiment) can selectively antibodies out a mixed population. The use of streptavidin or avidin coupled to a solid phase lends itself to this. The Cleaning can according to the invention but also on the basis of other affinity materials. The Elution of the antibodies is done by known standard methods.

Solution of the described technical issues

The solution to the technical problems described above is achieved by providing the embodiments characterized in the claims. The invention relates to the use of lumazine synthase molecules as a carrier protein for foreign proteins, peptides and / or other organic chemical molecules. Furthermore, the subject of this invention is a method for the selective, recombinant incorporation of the above-mentioned foreign proteins, or peptides in both loops or fiction preferably at the N-terminus and / or at the C-terminus of lumazine synthases. The method involves in vivo association of different lumazine synthase conjugates by co-expression of the genes of interest in a cell. Further, the method involves the possibility of in vitro reassociation of individually designed lumazine synthase conjugates into spherical particles by denaturation / renaturation of monomeric subunits, which can be performed both with and without the use of a folding assisting ligand.

Provided are lumazine synthase conjugates, which have a biotinylatable peptide (Tucker and Grisshammer, 1996; Schatz, 1993; Cronan, 1990) at the C-terminus feature. Furthermore, an artificial lumazine synthase molecule is provided, which at its C-terminus via an easily accessible basic amino acid (Lysine) features. Furthermore, a lumazine synthase molecule is provided which has a easily accessible cysteine molecule at the C-terminus. Both variants are suitable for the chemical coupling of organic Molecules. The coupling may e.g. done by making an acid amide bond or a disulfide bond between protein and coupling component. The chemical coupling according to the amide principle can also be carried out Lysine residues, naturally on the surface of lumazine synthase molecules occurrence.

Furthermore is a thermostable icosahedral lumazine synthase (from Aquifex aeolicus), which inter alia acts as a carrier protein for the preparation of particularly stable lumazine synthase conjugates suitable.

The method of manufacture of lumazine synthase conjugates includes the following sub-steps:

I. Preparation of the Fusion Vector

• A) Obtaining a DNA that is a gene for a lumazine synthase contains (e.g., by isolation from an organism, by PCR amplification with course occurring RNA or DNA as a matrix, or by DNA synthesis)
• B) Introduction suitable restriction sites for later insertion of foreign DNA into the lumazine synthase gene; Adaptation of the lumazine synthase sequence to special requirements using known molecular biological or biochemical mutagenesis methods; Insertion of DNA into a cloning vector using known molecular biology Methods. (Alternatively, the coding for the foreign peptide DNA section directly using the polymerase chain reaction and synthetic oligonucleotides fused to the lumazine synthase gene be guaranteed, with II.D guaranteed have to be)
• C) Transformation of host cells with the resulting plasmid
• D) Selection of transformants with the help of antibiotics or other selection method
• E) Analysis of transformants using molecular biological and biochemical methods, e.g. Restriction mapping, sequencing, Measurement of enzymatic activity, etc.

II. Insertion of a for a foreign peptide coding DNA

• A) Cloning of foreign DNA using molecular biological Methods or synthetic preparation of a DNA using chemical process
• B) Analysis of the DNA using molecular biological methods
• C) preparation the for the fusion foreign peptide-encoding DNA
• D) insertion of the prepared DNA to the 5 'and / or to the 3'-end and / or prepared in a loop region of the lumazine synthase gene in I. below Vector, so that one Fusion of the foreign gene with the lumazine synthase gene takes place. The Cloning must be done so that all reading frames used are inserted in the correct reading frame so that the fused gene fragments be translated together as a fusion protein.
• E) Transformation of host cells with the resulting plasmid
• F) Selection of transformants with the help of antibiotics or other selection method
• G) Analysis of the transformants using molecular biological and biochemical methods, e.g. Restriction mapping, sequencing, Measurement of enzymatic activity, etc.

III. Expression and purification the hybrid polypeptides

• A) Fermentation of the host strain with the fused one Artificial DNA using known microbiological methods.
• B) Expression of the fused artificial DNA in the transformed Host cells as chimeric Protein. Expression of the artificial DNA may be intentional Post-translational change of the chimeric protein in vivo, e.g. Phosphorylation, glycosylation, biotinylation, etc. include.
• C) preparation a cell extract with the fused polypeptide
• D) Purification of the fusion protein by means of chromatographic or other methods
• E) If necessary: solubilization and in vitro folding (Renaturation)
• F) If necessary: Chemical modification of the surface of Lumazine variants
• G) If necessary: In vitro association under combination different lumazine synthase variants

Further explanation of the procedure:

at Use of the present invention can be a variety of different Vectors are used. Extrachromosomal (episomal) vectors (e.g., plasmids), integration vectors (e.g., lambda vectors), Agrobacterium tumafaciens-based vectors for Plants (e.g., Ti plasmid). Plasmid vectors are preferred according to the invention. Used plasmids can from natural Sources isolated or synthetically manufactured. The selected plasmid should be compatible with the relevant host strain. This should be it u.a. above a suitable origin of replication appropriate to the host strain feature. Further should be the capacity the vector for both the used lumazine synthase variant as well as for the fused one Sufficient foreign peptide. Furthermore, on the plasmid vector singular Interfaces required for cloning DNA fragments. Of the Plasmid vector should be over suitable selection mechanisms, e.g. have a resistance gene. The Selection is necessary to distinguish host cells with or without plasmid to be able to.

If Escherichia coli is selected as host strain, according to the invention vectors, the one about Promoter sequence from bacteriophage T5 or T7, via a Operator sequence, preferably the operator sequence from Escherichia coli lactose operon (lacI), via a cloning cassette with multiple unique restriction endonuclease sites and an efficient termination sequence are preferred. Furthermore, should the vector over have an origin of replication, the high copy number of extrachromosomal DNA in the host cell allowed.

prokaryotic Expression systems are generally well suited to recombinant Production of Protein Conjugates According to the Invention In some cases can but posttranslational changes necessary, which are not introduced in prokaryotic organisms can. Eukaryote proteins can For example, in prokaryotes not glycosylated or phosphorylated become. Preferably, therefore, eukaryotic foreign proteins (fused to the lumazine synthase), which has such a post-translational Need modification, under the control of a strong promoter (e.g., AOX1) in low Eukaryotes (e.g., Pichia pastoris) or under the control of one for mammalian cells specific promoter (e.g., rat preproinsulin gene promoter) in mammalian cells (e.g., COS7 monkey kidney cells) or one for Insect cells (e.g., baculovirus, Autographa californica) specific Promoter (e.g., polyhedrin promoter). The vectors used should be compatible with these host strains.

to Provide oral plant-based vaccines e.g. Vector systems based on the Ti plasmid of Agrobacterium to tumefaciens. As an alternative to gene transfer to plants (e.g. in monocotyledonous plants, such as rice, wheat, maize, etc.) are suitable physical methods (e.g., microprojectile bombardment).

It can both natural occurring proteins as well as synthetic proteins found in the Nature does not occur to the carrier protein (Lumazine synthase) are fused. As DNA sources are e.g. Viruses, prokaryote (Eubacteria, Archaea) and eukaryotic organisms (plants, animals) in question. The DNA to be fused can also be synthetic, under Use for this more usual Methods to be produced. Furthermore, the DNA can also help Reverse transcriptase can be produced from mRNA.

The Recombinantly generated plasmid vectors become transformation used by host cells. According to the invention, preference is given to good characterization Bacterial cells. The host cells can also have eukaryotic cells be. Host strains used should over the enzymatic enzyme necessary for expression of the fused polypeptide Equipment. Transformation techniques are known in the art. Protocols are included Maniatis et al. (1982). After the transformation takes place an analysis of the transformants. The plasmids are isolated and using molecular biological methods, such as restriction analysis, DNA sequencing, characterized.

The Expression of a cloned DNA sequence in a prokaryotic or eukaryotic host cell is known in the art. The Cultivation of the transformed host cell in the method according to the invention for obtaining recombinant fusion proteins among those Conditions held for the Expression of the DNA sequence conducive are. The cell digestion after The expression can be carried out by all methods customary for this purpose. The digested cells are prepared using known separation techniques in a soluble and an insoluble one Separated fraction.

Should the fusion protein in the form of inclusion bodies in the insoluble Fraction, then the remaining after centrifugation cell residue washed and then by adding a solubilizing agent. The solubilization takes place preferably in the presence of reducing agents. The insoluble Substances are separated by known methods. The inventive method is characterized in that the Renaturation step in the presence of a stabilizing agent (5-nitro-6- (D-ribitylamino) 2,4 (1H, 3H) -pyrimidinedione) carried out can be.

The Purification of the fusion proteins can be performed by means of known chromatographic or other biochemical methods.

By the molecular biology introduction a reactive amino acid, According to the invention preferably one Lysine and / or a cysteine residue, coupled to a flexible Peptide linker, becomes a covalent coupling of molecules allows chemical routes or relieved. The coupling can according to the invention to different Species take place. Examples to be mentioned here are: a) bisimide esters are readily soluble in water and can under mild reaction conditions (pH 7.0-pH 10.0) with the ε-amino group react with a lysine residue. The resulting amide bond is stable. Activated lumazine synthases in this way can be coupled with others Peptides are used. b) carbodiimides belong to a linking group, which are to be described by the general formula R-N = C = N-R '. R and R 'can through aliphatic or aromatic radicals are represented. carbodiimides react preferentially with the ε-amino group from lysine. c) m-maleimido-benzoyl-N-hydroxysuccinimide ester (MBS) is a well studied heterobifunctional reagent. In neutral aqueous solution MBS reacts first in an acylation reaction with amino groups to the activated N-hydroxysuccinimide ester. A second peptide may then be added by addition of a thiol residue bound to the double bond of the ester. d) N-succinimidyl-3- (2-pyridyldithio) -propionate (SPDP) is a heterobifunctional reagent which is mild Conditions can react with amino groups of the target protein. The 2-Pyridyldisulfid structure can then with aliphatic thiols or a cysteine of another peptide via a thiol-disulfide exchange reaction react. The coupling reaction can take place in the pH range of 5-9 and the progress of the reaction can be monitored photometrically. There are no known reactions with other functional groups.

The inventive production in vitro reassoziierten Lumazinsynthase conjugates via a Dissociation step and a subsequent folding / reassoziationsschritt. The dissociation can be done by treatment with denaturing Agents, e.g. Urea or guanidine chloride, by amendment of the pH, by heat treatment or other procedures. The monomer present after denaturation chimeric Proteins each consist of a constant region, one Lumazine synthase (or a modified Lumazinsynthase) and a variable region (any fused peptide) together. The monomeric subunits can subsequently be mixed as desired. Since the recombined subunits respectively have a constant proportion of lumazine synthase is a renaturation the lumazine synthase core structure to form the natural icosahedral structure possible. The renaturation can be carried out in the presence of a stabilizing agent (Preferably, 5-nitro-6- (D-ribitylamino) 2,4 (1H, 3H) -pyrimidinedione) take place.

The In vivo combination of different lumazine synthase variants takes place by way of coexpression of the respective genes which are responsible for the desired fusion polypeptide encode. The genes in question can be found here on the chromosomal DNA of the host strain and / or on one or more plasmid vectors are located. By coordinating the expression of the in vivo to be associated Lumazine synthase variants can be a certain mixing ratio, i.e. set certain combination variants.

The invention further describes:
Protein consisting of the lumazine synthase from Bacillus subtilis, characterized in that the amino acid cysteine at position 93 is replaced by the amino acid serine.
Protein consisting of the lumazine synthase from Bacillus subtilis, characterized in that the amino acid cysteine at position 139 is replaced by the amino acid serine.
Protein consisting of the lumazine synthase from Bacillus subtilis, characterized in that the Aminosäu re cysteine at positions 93 and 139 are replaced by the amino acids serine.
DNA adapted to the codon usage of Escherichia coli for the production of the lumazine synthase from Aquifex aeolicus in a recombinant Escherichia coli strain.
Protein consisting of the lumazine synthase from Aquifex aeolicus for use as a carrier protein according to claim 1.
Chimeric protein consisting of the amino acids 1-60 of the lumazine synthase from Bacillus subtilis and the amino acids 61-154 of the lumazine synthase from Aquifex aeolicus for use as a carrier protein according to claim 1.

Use of the protein conjugates according to the invention for the manufacture of a medicament or a vaccine.

In the drawings mean:

1 a schematic representation of an ELISA protocol for the detection of a particular antigen or a specific antibody. The antigen is bound to the microtiter plate. The enzyme (E) is coupled to the secondary antibody. The colorless substrate is converted by the enzyme (E) to a colored product.

2 describes the detection of an antigen by means of a biotin-labeled primary antibody. A lumazine synthase conjugate (amplifying linker molecule) containing up to 60 biotin molecules covalently bound is bound to the biotinylated primary antibody via a streptavidin or an avidin bridge (SA). The color reaction is achieved by any streptavidin-coupled enzyme (E) which complexes with the biotinylated lumazine synthase. The interposition of a 60-fold biotinylated linker protein (lumazine synthase conjugate) and the resulting multiple binding of color-reaction-mediating enzyme, an extreme signal amplification is achieved, the signal strength is proportional to the antigen concentration.

• A) Ein Lumazinsynthasemolekül, welches 1–5 kurze Peptide von antigen wirksamen viralen oder bakteriellen Oberflächenproteinen (antigene Determinanten) und bis zu 60 Biotinmoleküle kovalent gebunden enthält, dient als Detektionsmolekül für immobilisierte Antikörper, die aus einem Patientenserum oder anderen Flüssigkeiten stammen.
• B) Charakteristische Antikörper gegen bestimmte Infektionskrankheiten werden mit Hilfe spezieller immobilisierter Epitope (Teile von Oberfächenproteinen der betreffenden pathogenen Organismen; antigene Determinanten) aus der jeweiligen Körperflüssigkeit gefischt. Ein Lumazinsynthasemolekül, welches ebenfalls 1–5 Kopien des oben bezeichneten Epitops und bis zu 60 Biotinmoleküle kovalent gebunden enthält, dient als Detektionsmolekül für diese nun immobilisierten Antikörper.

3 describes the use of a lumazine synthase mixed conjugate in the diagnosis of infectious diseases.

• A) A lumazine synthase molecule containing 1-5 short peptides of antigenically active viral or bacterial surface proteins (antigenic determinants) and up to 60 biotin molecules covalently bound serves as a detection molecule for immobilized antibodies derived from a patient's serum or other fluids.
• B) Characteristic antibodies against certain infectious diseases are fished out of the respective body fluid with the help of special immobilized epitopes (parts of surface proteins of the respective pathogenic organisms, antigenic determinants). A lumazine synthase molecule, which also contains 1-5 copies of the above-identified epitope and up to 60 biotin molecules covalently bound, serves as a detection molecule for these now immobilized antibodies.

A Color reaction is in both cases by any streptavidin-coupled enzyme (E), which reaches a complex with the biotinylated lumazine synthase. Through the interposition of a multiply biotinylated linker protein (Lumazine synthase conjugate) and the resulting multiple Binding of color reaction-mediating enzyme, signal amplification is achieved.

Not bound antibodies are removed in a first washing step. Does not bind to the immobilized epitopes, so does the complex of lumazine synthase conjugate and antibodies not formed. Excess lumazine synthase conjugate is removed in a second washing step, so that the test batch remains colorless.

4 describes a schematic representation of a test arrangement for the purification of antibodies with a specific antigen recognition. A lumazine synthase conjugate which has both a desired foreign peptide (in multiple execution) and a biotinylated portion (in a simple embodiment) is bound to its immobilized streptavidin via its biotin moiety. The streptavidin molecules are coupled to a solid phase. The antibody mix population is placed on a column of immobilized streptavidin (or mixed with streptavidin material) with the antibodies of the desired specificity binding to the foreign peptide portion on the lumazine synthase conjugate.

Of the Washing process of Streptavidin-lumazine synthase conjugate-antibody complex and subsequent elution the specific antibody is done by known standard methods.

5 shows a schematic representation of the structure of a biosensor, which may in principle consist of at least three parts: 1. The biological receptor, 2. The transducer unit, 3. The affiliated electronics. The biological receptor can be bound to the transducer or transductor in different ways.

6 schematically shows a functionalized lumazine synthase with 60 identical or differently modified subunits, which is connected via an anchor (peptide, fatty acid, other functional groups) with a surface (for example transducer surface, membrane, other surfaces, etc.). The detection sensitivity for foreign-molecule binding on the surface of the lumazine synthase is increased by a high number of functional groups (for example epitopes for antibody recognition, antibodies for the detection of foreign molecules in solution or other receptors).

7 shows schematically a possible construction of a field effect transistor involving a multimeric, functionalized lumazine synthase. A change in the surface charge at the gate electrode, which occurs through the binding of a foreign molecule to the surface of the lumazine synthase, thereby modulating the flow of current through the field effect transistor.

8th shows a sequence comparison of lumazine synthases from the following organisms: 1. Mycobacterium avium; 2. Mycobacterium tuberculosis; 3. Corynebacterium ammoniagenes; 4. Chlorobium tepidum; 5. Aquifex aeolicus; 6. Thermotoga maritima; 7. Bacillus subtilis; 8. Bacillus amyloliquefaciens; 9. A. pleuropneumoniae; 10. Streptococcus pneumoniae; 11. Staphylococcus aureus; 12. Vibrio cholerae; 13. Photobacterium phosporeum; 14. S. putrefaciens; 15. Photobacterium leiognathi; 16. Shigella flexneri; 17. Escherichia coli; 18. Haemophilus influenzae; 19. Dehalospirillum multivorans; 20. Helicobacter pylori; 21. Deinococcus radiodurans; 22. Synechocystis sp .; 23. Porphyromonas gingivalis; 24. Arabidopsis thaliana; 25. Methanococcus jannaschii; 26. Archaeoglobus fulgidus; 27. Methanobacterium thermoautotrophicum; 28. Chlamydia trachomutis; 29. Saccharomyces cerevisiae; 30. Brucella abortus. The protein sequences were obtained by translation of the underlying DNA sequences. The sequence set shown was obtained by database search using the search algorithms of Altschul et al. (1997) and the sequence of lumazine synthase from Bacillus subtilis as a search template.

9 shows the top view of a pentameric subunit of the icosahedral lumazine synthase from Bacillus subtilis. The ligand 5-nitro-6- (D-ribitylamino) -2,4- (1H, 3H) -pyrimidinedione binds at the point of contact between two monomeric subunits (Ladenstein et al., 1988, 1994, Ritsert et al., 1995). ,

10 shows a model of the icosahedral lumazine synthase from Bacillus subtilis. One of 12 pentameric subunits is highlighted by the use of different shades of gray. Both the N- and the C-terminus lie on the surface and are freely accessible.

11 shows the expression vectors used in the embodiments. SD, ribosomal binding site; MCS, cloning cassette with singular interfaces; t ₀ , t ₁ , terminator sequences; (cat), non-active gene for chloramphenicol acetyltransferase (shifted reading frame); (Δcat), inactive gene for chloramphenicol acetyltransferase (deletion); Restriction sites are indicated by letters: B, BamHI; E, EcoRI; H, HindIII; N, NcoI; P, PstI; S, SalI. Translation start in vector pNCO113 at position 113 and at position 233 at p602 / -CAT.

12 describes the 1st PCR for the introduction of a mutation using the example of the replacement of the amino acid cysteine at position 93 with serine in the gene of the lumazine synthase from Bacillus subtilis. In the first step of directed mutagenesis, two separate PCR approaches, with the oligonucleotide pairings PNCO-M1 / C93S and PNCO-M2 / RibH-3 and the expression plasmid pNCO-BS-LuSy as template, are performed first. Fragment A contains the intended mutation and an intact recognition sequence for the restriction endonuclease EcoRI. Fragment B represents the entire but unmutated ribH gene (lumazine synthase from Bacillus subtilis). In this fragment, the 5 'cloning site is deleted. (R: ribosomal binding site)

13 describes the 2nd PCR in the introduction of a mutation. In the second step of mutagenesis, the introduced mutation, which is now still at the end of the 3'-end of the gene fragment generated by PCR, is introduced by overlapping extension into the entire gene.

14 describes the 3rd PCR for introducing a mutation. The third PCR serves to increase the elongated codon strand of fragment A.

In the 15 to 24 . 26 to 28 and 30 and 31 the sequence of each lumazine synthase used is indicated by bold type. The linker regions are underlined. Recognition sequences for the respective restriction endonucleases are in italics and underlined. Fused sequences, or amino acids that are not counted to the linker sequence, are underlined dotted. The amino acid sequence is given in single letter code.

15 shows the structure of the pNCO-N-BS-LuSy vector for the fusion of foreign proteins to the N-terminus of lumazine synthase.

16 shows the structure of the vector pNCO-C-BS-LuSy for the fusion of foreign proteins to the C-terminus of lumazine synthase.

17 shows the structure of the vector pNCO-BS-LuSy-EC-DHFR.

18 shows the structure of the vector pNCO-N-VP2-BS-LuSy in the region of the N-terminus.

19 shows the structure of the vector pNCO-C-V2-BS-LuSy in the region of the C-terminus.

20 shows the structure of the vector pNCO-C-Biotag-BS-LuSy in the region of the C-terminus.

21 show the structure of the vector pNCO-Lys165-BS-LuSy in the region of the C-terminus.

22 shows the structure of the vector pNCO-Cys167-RS-LuSy in the region of the C-terminus.

23 shows the structure of the vector pFLAG-MAC-BS-LuSy in the region of the N-terminus.

24 shows the structure of the vector pNCO-C-His6-BS-LuSy in the region of the C-terminus.

25 Figure 4 shows the construction of Aquifex aeolicus thermostable lumazine synthase (Deckert et al., 1998) using 11 synthetic oligonucleotides (AQUI-1 through AQUI-11) and a 6-step polymerase chain reaction.

26 shows the coupling of an artificial 13 amino acid in vivo biotinylatable peptide to the C-terminus of the thermostable lumazine synthase from Aquifex aeolicus. (Peptide is linked via a linker with 3 alanine residues to the C-terminus of the carrier protein)

27 shows the coupling of an artificial 13 amino acid in vivo biotinylatable peptide via a linker of 6 histidine and 3 alanine residues to the C-terminus of the thermostable lumazine synthase from Aquifex aeolicus.

28 shows the coupling of an artificial 13 amino acid in vivo biotinylatable peptide, via a linker consisting of 6 histidine residues and the sequence Gly-Gly-Ser-Gly-Ala-Ala-Ala to the C-terminus of the thermostable lumazine synthase from Aquifex aeolicus.

29 shows the production of a chimeric protein consisting of a part of the lumazine synthase from Bacillus subtilis and part of the thermostable lumazine synthase from Aquifex aeolicus. (RBS: ribosomal binding site)

30 shows the 5 'region of the vector pNCO-AA-BglII-LuSy or the vector pNCO-AA-BglII-LuSy- (BamHI) for the fusion of foreign genes to the 5' end of the lumazine synthase from Aquifex aeolicus. The newly introduced into the sequence recognition sequence for the unique restriction endonuclease BglII is shown.

31 shows the 3 'region of the vector pNCO-AA-LuSy (BamHI) or the vector pNCO-AA-BglII-LuSy (BamHI) for the fusion of foreign genes to the 3' end of the lumazine synthase from Aquifex aeolicus. At the C-terminus of the carrier protein, a peptide having the sequence GSVDLQPSLIS is fused.

• SEQ ID No.1 zeigt die DNA-Sequenz des Expressionsvektors pNCO113. (Vektor zur Expression von Genen in Escherichia coli; Stüber et al., 1990)
• SEQ ID No.2 zeigt die DNA-Sequenz des Expressionsvektors p602/-CAT. (Schaukelvektor zur Expression von Genen in Escherichia coli und Bacillus subtilis; Henner, 1990; LeGrice, 1990)
• SEQ ID No.3 zeigt die DNA-Sequenz des Expressionsplasmides pNCO-BS-LuSy. (Expressionsplasmid mit der unveränderten Lumazinsynthase aus Bacillus subtilis zur Expression in Escherichia coli)
• SEQ ID No.4 zeigt die DNA-Sequenz des Expressionsplasmides p602-BS-LuSy. (Expressionsplasmid mit der unveränderten Lumazinsynthase aus Bacillus subtilis zur Expression in Escherichia coli und Bacillus subtilis)
• SEQ ID No.5 zeigt die DNA-Sequenz des Expressionsplasmides pNCO-BS-LuSy-C93S. (Expressionsplasmid mit einer veränderten Lumazinsynthase-Variante, wobei die Aminosäure Cystein an Position 93 durch die Aminosäure Serin ausgetauscht wurde)
• SEQ ID No.6 zeigt die DNA-Sequenz des Expressionsplasmides pNCO-BS-LuSy-C139S. (Expressionsplasmid mit einer veränderten Lumazinsynthase-Variante, wobei die Aminosäure Cystein an Position 139 durch die Aminosäure Serin ausgetauscht wurde)
• SEQ ID No.7 zeigt die DNA-Sequenz des Expressionsplasmides pNCO-BS-LuSy-C93/134S. (Expressionsplasmid mit einer veränderten Lumazinsynthase-Variante, wobei die Aminosäure Cystein an den Positionen 93 und 139 durch die Aminosäure Serin ausgetauscht wurde)
• SEQ ID No.8 zeigt die DNA-Sequenz des Expressionsvektors pNCO-N-BS-LuSy zur Fusion von Fremdpeptiden an den N-Terminus der Lumazinsynthase aus Bacillus subtilis.
• SEQ ID No.9 zeigt die DNA-Sequenz des Expressionsvektors pNCO-C-BS-LuSy zur Fusion von Fremdpeptiden an den C-Terminus der Lumazinsynthase aus Bacillus subtilis.
• SEQ ID No.10 zeigt die DNA-Sequenz des Expressionsvektors pNCO-EC-DHFR-BS-LuSy. (Expressionsplasmid zur Expression eines Fusionsproteins, bestehend aus der Dihydrofolatreduktase aus Escherichia coli und der Lumazinsynthase aus Bacillus subtilis, wobei die Dihydrofolatreduktase an den N-Terminus der Lumazinsynthase fusioniert vorliegt)
• SEQ ID No.11 zeigt die DNA-Sequenz des Expressionsvektors pNCO-EC-MBP-BS-LuSy. (Expressionsplasmid zur Expression eines Fusionsproteins, bestehend aus dem maltosebindenden Protein aus Escherichia coli und der Lumazinsynthase aus Bacillus subtilis, wobei das maltosebindene Protein an den N-Terminus der Lumazinsynthase fusioniert vorliegt)
• SEQ ID No.12 zeigt die DNA-Sequenz des Expressionsvektors pNCO-BS-LuSy-EC-DHFR. Die Linkersequenz zwischen der Lumazinsynthase und der Dihydrofolatreduktase ist punktiert unterstrichen. (Expressionsplasmid zur Expression eines Fusionsproteins, bestehend aus der Dihydrofolatreduktase aus Escherichia coli und der Lumazinsynthase aus Bacillus subtilis, wobei die Dihydrofolatreduktase an den C-Terminus der Lumazinsynthase fusioniert vorliegt)
• SEQ ID No.13 zeigt die DNA-Sequenz des Expressionsvektors pNCO-N-VP2-BS-LuSy. (Expressionsplasmid zur Expression eines Fusionsproteins, bestehend aus der VP2-Domäne des 'Minc enteritis Virus' und der Lumazinsynthase aus Bacillus subtilis, wobei sich die VP2-Domäne am N-Terminus befindet; das ehemalige Startcodon der Lumazinsynthase ist unterstrichen)
• SEQ ID No.14 zeigt die DNA-Sequenz des Expressionsvektors pNCO-C-VP2-BS-LuSy. (Expressionsplasmid zur Expression eines Fusionsproteins, bestehend aus der VP2-Domäne des 'Mine enteritis Virus' und der Lumazinsynthase aus Bacillus subtilis, wobei sich die VP2-Domäne am C-Terminus befindet)
• SEQ ID No.15 zeigt die DNA-Sequenz des Expressionsvektors pNCO-N/C-VP2-BS-LuSy. (Expressionsplasmid zur Expression eines Fusionsproteins, bestehend aus der VP2-Domäne des 'Minc enteritis Virus' und der Lumazinsynthase aus Bacillus subtilis, wobei sich die VP2-Domäne sowohl am N-Terminus als auch am C-Terminus befindet; das ehemalige Startcodon der Lumazinsynthase ist unterstrichen)
• SEQ ID No.16 zeigt die DNA-Sequenz des Expressionsvektors pNCO-C-Biotag-BS-LuSy. (Expressionsplasmid zur Expression eines Fusionsproteins, bestehend aus einem 13 Aminosäuren langen, in vivo biotinylierfähigen Peptid und der Lumazinsynthase aus Bacillus subtilis, wobei sich das fusionierte Peptid am C-Terminus befindet)
• SEQ ID No.17 zeigt die DNA-Sequenz des Expressionsvektors pNCO-Lys165-BS-LuSy. (Expressionsplasmid zur Expression einer veränderten Lumazinsynthase aus Bacillus subtilis, bei der der C-Terminus verlängert wurde und mit einem Lysinrest endet; das Codon für Lysin (AAA) ist unterstrichen)
• SEQ ID No.18 zeigt die DNA-Sequenz des Expressionsvektors pNCO-Cys167-BS-LuSy. (Expressionsplasmid zur Expression einer veränderten Lumazinsynthase aus Bacillus subtilis, bei der der C-Terminus verlängert wurde und mit einem Cysteinrest endet; das Codon für Cystein (TGC) ist unterstrichen)
• SEQ ID No.19 zeigt die DNA-Sequenz des Expressionsvektors pFLAG-MAC-BS-LuSy. (Expressionsplasmid zur Expression eines Fusionsproteins, bestehend aus einem 12 Aminosäuren langen Epitop, welches von einem monoklonalen Antikörper erkannt wird, und der Lumazinsynthase aus Bacillus subtilis, wobei sich das fusionierte Peptid am N-Terminus befindet; das ehemalige Startcodon der Lumazinsynthase ist unterstrichen)
• SEQ ID No.20 zeigt die DNA-Sequenz des Expressionsvektors pNCO-C-His6-BS-LuSy. (Expressionsplasmid zur Expression eines Fusionsproteins, bestehend aus einem 6 Aminosäuren langen Peptid (6 × Histidin) und der Lumazinsynthase aus Bacillus subtilis, wobei sich das fusionierte Peptid am C-Terminus befindet; das Peptid ist unterstrichen)
• SEQ ID No.21 zeigt die DNA-Sequenz des Expressionsvektors pNCO-AA-LuSy. (Expressionsplasmid zur Expression der unveränderten, thermostabilen Lumazinsynthase aus Aquifex aeolicus; die DNA-Sequenz wurde an die Codon-Verwendung von Escherichia coli angepasst; die DNA wurde vollsynthetisch hergestellt)
• SEQ ID No.22 zeigt die DNA-Sequenz des Expressionsvektors pNCO-C-Biotag-AA-LuSy. (Expressionsplasmid zur Expression eines Fusionsproteins, bestehend aus einem 13 Aminosäuren langen, in vivo biotinylierfähigen Peptid und der Lumazinsynthase aus Aquifex aeolicus, wobei sich das fusionierte Peptid am C-Terminus befindet; das Peptid ist über einen Linker mit 3 Alaninresten mit dem C-Terminus des Trägerproteins verbunden)
• SEQ ID No.23 zeigt die DNA-Sequenz des Expressionsvektors pNCO-His6-C-Biotag-AA-Lusy. (Expressionsplasmid zur Expression der Lumazinsynthase aus Aquifex aeolicus mit C- terminal, über einen Linker aus 6 Histidin- und 3 Alaninresten gekoppeltem, in vivo biotinylierfähigem Peptid)
• SEQ ID No.24 zeigt die DNA-Sequenz des Expressionsvektors pNCO-His6-GLY2-SER-GLY-C-Biotag-AA-LuSy. (Expressionsplasmid zur Expression der Lumazinsynthase aus Aquifex aeolicus mit C-terminal, über einen Linker bestehend aus der Aminosäurenfolge IHGGSGAAA gekoppeltem, in vivo biotinylierfähigem Peptid)
• SEQ ID No.25 zeigt die DNA-Sequenz des Expressionsvektors pNCO-BS-LuSy-AgeI-AA-Lusy. (Expressionsplasmid zur Expression eines chimären Proteins bestehend aus einem Teil Lumazinsynthase aus Bacillus subtilis und einem Teil der thermostabilen Lumazinsynthase aus Aquifex aeolicus; der Anteil der Lumazinsynthase aus Bacillus subtilis ist fettgedruckt, der Anteil der Lumazinsynthase aus Aquifex aeolicus ist doppelt unterstrichen)
• SEQ ID No.26 zeigt die DNA-Sequenz des Expressionsvektors pNCO-AA-BglII-LuSy. (Vektor zur Fusion von Fremdpeptiden an den N-Terminus bzw. an das 5'-Ende der thermostabilen Lumazinsynthase aus Aquifex aeolicus unter Verwendung der Restriktionsendonuklease BglII).
• SEQ ID No.27 zeigt die DNA-Sequenz des Expressionsvektors pNCO-AA-LuSy-(BamHI) (Vektor zur Fusion von Fremdpeptiden an den C-Terminus bzw. an das 3'-Ende der thermostabilen Lumazinsynthase aus Aquifex aeolicus unter Verwendung der Restriktionsendonuklease BamHI).
• SEQ ID No.28 zeigt die DNA-Sequenz des Expressionsvektors pNCO-AA-BglII-LuSy-(BamHI). (Vektor zur Fusion von Fremdpeptiden an den N- und an den C-Terminus bzw. an das 5'- und an das 3'-Ende der thermostabilen Lumazinsynthase aus Aquifex aeolicus unter Verwendung der Restriktionsendonukleasen BglII und BamHI)

The listed DNA sequence protocols explain the structure of the plasmids listed in the examples. In the Sequence Listings, the recognition sequences of the particular restriction endonucleases used are italicized and underlined; the expressed fusion proteins are each in bold and Linker sequences are punctuated underlined; Exceptions in character formatting are noted.

• SEQ ID No.1 shows the DNA sequence of the expression vector pNCO113. (Vector for expression of genes in Escherichia coli, Stüber et al., 1990)
• SEQ ID No.2 shows the DNA sequence of the expression vector p602 / -CAT. (Swing vector for the expression of genes in Escherichia coli and Bacillus subtilis, Henner, 1990; LeGrice, 1990)
• SEQ ID No.3 shows the DNA sequence of the expression plasmid pNCO-BS-LuSy. (Expression plasmid with the unchanged lumazine synthase from Bacillus subtilis for expression in Escherichia coli)
• SEQ ID No.4 shows the DNA sequence of the expression plasmid p602-BS-LuSy. (Expression plasmid with the unchanged lumazine synthase from Bacillus subtilis for expression in Escherichia coli and Bacillus subtilis)
• SEQ ID No.5 shows the DNA sequence of the expression plasmid pNCO-BS-LuSy-C93S. (Expression plasmid with a modified lumazine synthase variant, wherein the amino acid cysteine at position 93 has been replaced by the amino acid serine)
• SEQ ID No.6 shows the DNA sequence of the expression plasmid pNCO-BS-LuSy-C139S. (Expression plasmid with a modified lumazine synthase variant, wherein the amino acid cysteine at position 139 has been replaced by the amino acid serine)
• SEQ ID No.7 shows the DNA sequence of the expression plasmid pNCO-BS-LuSy-C93 / 134S. (Expression plasmid with a modified lumazine synthase variant, wherein the amino acid cysteine at positions 93 and 139 has been replaced by the amino acid serine)
• SEQ ID No. 8 shows the DNA sequence of the expression vector pNCO-N-BS-LuSy for the fusion of foreign peptides to the N-terminus of the lumazine synthase from Bacillus subtilis.
• SEQ ID No. 9 shows the DNA sequence of the expression vector pNCO-C-BS-LuSy for the fusion of foreign peptides to the C-terminus of the lumazine synthase from Bacillus subtilis.
• SEQ ID No.10 shows the DNA sequence of the expression vector pNCO-EC-DHFR-BS-LuSy. (Expression plasmid for expression of a fusion protein consisting of the dihydrofolate reductase from Escherichia coli and the lumazine synthase from Bacillus subtilis, wherein the dihydrofolate reductase is fused to the N-terminus of the lumazine synthase)
• SEQ ID No.11 shows the DNA sequence of the expression vector pNCO-EC-MBP-BS-LuSy. (Expression plasmid for expressing a fusion protein consisting of the maltose binding protein from Escherichia coli and the lumazine synthase from Bacillus subtilis, wherein the maltose binding protein is fused to the N-terminus of lumazine synthase)
• SEQ ID No.12 shows the DNA sequence of the expression vector pNCO-BS-LuSy-EC-DHFR. The linker sequence between the lumazine synthase and the dihydrofolate reductase is punctuated underlined. (Expression plasmid for expression of a fusion protein consisting of the dihydrofolate reductase from Escherichia coli and the lumazine synthase from Bacillus subtilis, wherein the dihydrofolate reductase is fused to the C-terminus of the lumazine synthase)
• SEQ ID No.13 shows the DNA sequence of the expression vector pNCO-N-VP2-BS-LuSy. (Expression plasmid for expression of a fusion protein consisting of the VP2 domain of the 'Minc enteritis virus' and the lumazine synthase from Bacillus subtilis, where the VP2 domain is located at the N-terminus, the former start codon of the lumazine synthase is underlined)
• SEQ ID No.14 shows the DNA sequence of the expression vector pNCO-C-VP2-BS-LuSy. (Expression plasmid for expressing a fusion protein consisting of the VP2 domain of the 'mine enteritis virus' and the lumazine synthase from Bacillus subtilis, wherein the VP2 domain is located at the C-terminus)
• SEQ ID No.15 shows the DNA sequence of the expression vector pNCO-N / C-VP2-BS-LuSy. (Expression plasmid for expression of a fusion protein consisting of the VP2 domain of the 'Minc enteritis virus' and the lumazine synthase from Bacillus subtilis, wherein the VP2 domain is located at both the N-terminus and the C-terminus, the former start codon of lumazine synthase is underlined)
• SEQ ID No.16 shows the DNA sequence of the expression vector pNCO-C biotag BS-LuSy. (Expression plasmid for expression of a fusion protein consisting of a 13 amino acid in vivo biotinylatable peptide and the lumazine synthase from Bacillus subtilis with the fused peptide at the C-terminus)
• SEQ ID No.17 shows the DNA sequence of the expression vector pNCO-Lys165-BS-LuSy. (Expression plasmid for expression of an altered lumazine synthase from Bacillus subtilis, in which the C-terminus has been extended and ends with a lysine residue, the codon for lysine (AAA) is underlined)
• SEQ ID No.18 shows the DNA sequence of the expression vector pNCO-Cys167-BS-LuSy. (Expression plasmid for expression of an altered lumazine synthase from Bacillus subtilis in which the C-terminus has been extended and ends with a cysteine residue, the codon for cysteine (TGC) is underlined)
• SEQ ID No.19 shows the DNA sequence of the expression vector pFLAG-MAC-BS-LuSy. (Expression plasmid for expression of a fusion protein consisting of a 12-amino acid epitope recognized by a monoclonal antibody and the lumazine synthase from Bacillus subtilis with the fused peptide at the N-terminus, the former start codon of lumazine synthase underlined)
• SEQ ID No.20 shows the DNA sequence of the expression vector pNCO-C-His6-BS-LuSy. (Expression plasmid for expression of a fusion protein consisting of a 6-amino acid peptide (6 × histidine) and the lumazine synthase from Bacillus subtilis, with the fused peptide at the C-terminus, the peptide is underlined)
• SEQ ID No.21 shows the DNA sequence of the expression vector pNCO-AA-LuSy. (Expression plasmid for expression of the unchanged, thermostable lumazine synthase from Aquifex aeolicus, the DNA sequence was adapted to the codon usage of Escherichia coli, the DNA was prepared fully synthetic)
• SEQ ID No.22 shows the DNA sequence of the expression vector pNCO-C Biotag-AA-LuSy. (Expression plasmid for expression of a fusion protein consisting of a 13-amino acid in vivo biotinylatable peptide and the lumazine synthase from Aquifex aeolicus with the fused peptide at the C-terminus; the peptide is linked to the C-terminus via a 3-alanine residue linker linked to the carrier protein)
• SEQ ID No.23 shows the DNA sequence of the expression vector pNCO-His6-C Biotag-AA-Lusy. (Expression plasmid for expression of the lumazine synthase from Aquifex aeolicus with C-terminal, via a linker of 6 histidine and 3 alanine residues coupled in vivo biotinylierfähigem peptide)
• SEQ ID No.24 shows the DNA sequence of the expression vector pNCO-His6-GLY2-SER-GLY-C-Biotag-AA-LuSy. (Expression plasmid for expression of the lumazine synthase from Aquifex aeolicus with C-terminal, via a linker consisting of the amino acid sequence IHGGSGAAA coupled, in vivo biotinylierfähigem peptide)
• SEQ ID No.25 shows the DNA sequence of the expression vector pNCO-BS-LuSy-AgeI-AA-Lusy. (Expression plasmid for expression of a chimeric protein consisting of a part of lumazine synthase from Bacillus subtilis and a part of the thermostable lumazine synthase from Aquifex aeolicus, the proportion of lumazine synthase from Bacillus subtilis is in bold, the proportion of lumazine synthase from Aquifex aeolicus is underlined twice)
• SEQ ID No.26 shows the DNA sequence of the expression vector pNCO-AA-BglII-LuSy. (Vector for the fusion of foreign peptides to the N-terminus or to the 5 'end of the thermostable lumazine synthase from Aquifex aeolicus using the restriction endonuclease BglII).
• SEQ ID No.27 shows the DNA sequence of the expression vector pNCO-AA-LuSy- (BamHI) (vector for the fusion of foreign peptides to the C-terminus or to the 3 'end of the thermostable lumazine synthase from Aquifex aeolicus using the restriction endonuclease Bam).
• SEQ ID No.28 shows the DNA sequence of the expression vector pNCO-AA-BglII-LuSy (BamHI). (Vector for the fusion of foreign peptides to the N- and the C-terminus or to the 5 'and to the 3' end of the thermostable lumazine synthase from Aquifex aeolicus using the restriction endonucleases BglII and BamHI)

The used bacterial strains become with the DSM according to Budapester Contract deposited. The deposit numbers will be submitted later.

in the The invention will be explained in more detail below with reference to examples.

The Concentration data "M, mM and μM "refer to Mol / l, mmol / l and μ mol / l ..

The in the embodiments used strains of Bacillus subtilis and Escherichia coli are shown below Genotype and reference listed.

The oligonucleotides used were from Gibco BRL, Eggenstein and MWG Biotech, Ebersberg produced as part of a custom synthesis.

example 1

Heterologous expression of the gene of lumazine synthase from Bacillus subtilis in Escherichia coli XL1

• A) The gene for the lumazine synthase from Bacillus subtilis was mixed with the oligonucleotides RibH-1 (5 'gag gag aaa tta acc atatatc ata caa gga aat tta g 3'), which at the 3 'end to the 5' end of the Lumazine synthase gene and encodes a portion of an optimized ribosome binding site at the 5 'end, and RibH-2 (5' tat did gga tcc cca tgg tta ttc gaa aga acg gtt taa gtt tg 3 ') which is at the 3' end is complementary to the gene of the lumazine synthase and introduces a recognition sequence for the endonuclease BamHI (G * GATCC) behind the stop codon, from the plasmid pRF2 (Perkins et al., 1991), which contains the entire RIB operon from Bacillus subtilis amplified (Mullis et al., 1986). 10 μl of PCR buffer (75 mM Tris / HCl, pH 9.0, 20 mM (NH ₄ ) ₂ SO ₄ , 0.01% (w / v) Tween 20) 6 μl Mg ²⁺ [1.5 mM] 8 μl dNTP's [ 200 μM each] 1 μl RibH-1 [0.5 μM] 1 μl RibH-2 [0.5 μM] 1 μl pRF2 [10 ng] 1 μl Goldstar Taq polymerase [0.5 U] (Eurogentec, Seraing , Belgium) 72 ul H ₂ O _bidest The batch was in the thermocycler (GeneAmp ^® PCR system 2400; Perkin Elmer) under the following conditions: 1. 95 ° C 5.0 min 0.5 min 2. 94 ° C 3. 0 , 5 min 50 ° C 4. 0.8 min 72 ° C 5. 7.0 now 72 ° C 6. cool down to 4 ° C Steps 2.-4. were repeated 20 times.
• B) The PCR batch was separated by agarose gel electrophoresis, the DNA visualized by staining with ethidium bromide under UV light, and the 498 bp fragment excised from the gel using a scalpel. The gel piece was weighed and added with 5 times the amount of 6M NaI solution. After melting the gel piece, glass milk (Geneclean II Kit, Bio101, San Diego, Calif.) Was added to this solution (2 μl of glass milk per 1 μg of DNA). After incubation on ice for 30 min, the suspension was centrifuged (Eppendorf centrifuge, 8000 rpm ^-1 , 2 min, 4 ° C.), the supernatant was discarded and the glass milk was suspended with 500 μl of NEW washing solution (composition not specified by the manufacturer), centrifuged (see above) ) and the supernatant then completely removed. The elution of the DNA was carried out with 30 μl bidistilled water (45 ° C, 15 min). The determination of the DNA concentration was carried out fluorometrically using the fluorescent dye Bisbenzimid H 33258 (Höchst, Frankfurt). In the presence of DNA, the absorption maximum of this dye at 365 nm, the emission maximum at 458 nm. The fluorometer used uses a mercury lamp and uses for excitation a wavelength of 365 nm in a bandwidth of 100 nm. The measurement of the emission by means of a photomultiplier at 460 nm in a bandwidth of 10 nm. Replication was carried out with 2 ml of TNE buffer (100 mM Tris / HCl pH 7.4, 10 mM EDTA, 1 M NaCl) containing 0.1 μg / ml of dye. Subsequently, 2 .mu.l of a calibration standard with known DNA concentration (plasmid DNA) were added and this concentration was adjusted on the fluorometer. After checking the zero point of the device with buffer / dye solution, 2 .mu.l of the DNA sample of unknown concentration were added and the concentration was read in .mu.g / .mu.l.
• C) 10 ng of the isolated DNA from B) then served as template for a second polymerase chain reaction with the oligonucleotides EcoRI-RBS-1 (5 'ata ata gaa ttc att aaa gag gag aaa tta acc atg 3'), which is the ribosomal binding site and immediately before the ribosomal binding site introduces a recognition sequence for the restriction endonuclease EcoRI (G * AATTC) into the DNA fragment, and RibH-2: 10 μl PCR buffer 6 μl Mg ²⁺ [1.5 mM] 8 μl dNTP's [ 200 μM each] 1 μl EcoRI-RBS [0.5 μM] 1 μl RibH-2 [0.5 μM] 1 μl DNA from the 1st PCR [10 ng] 1 μl Goldstar Taq Polymerase [0.5 μM] ] (Eurogentec, Seraing, Belgium) 72 μl H ₂ O _bidist The reaction was in a thermocycler (GeneAmp ^® PCR System 2400 Perkin Elmer) under the following conditions: 1. 95 ° C 5.0 min 0.5 min 2. 94 ° C 3. 0.5 ° C 4. 0 min 50 , 8 min 72 ° C 5. 7.0 min 72 ° C 6. cool down to 4 ° C The steps 2.-4. were repeated 30 times.
• D) The PCR batch was separated by agarose gel electrophoresis and the fragment with a length of 516 bp as described under B).
• E) The isolated DNA fragment was digested with the restriction endonucleases EcoRI and BamHI. 30.0 μl fragment from D) 2.5 μl EcoRI [62.5 U] 3.0 μl BamHI [60 U] 24.0 μl OPAU (10 ×; 500 mM potassium acetate; 100 mM magnesium acetate; 100 mM; Acetate, pH 7.5) 60.5 μl H ₂ O _bidist. The restriction enzymes were obtained from Pharmacia Biotech (Freiburg). The batch was incubated for 180 min at 37 ° C. and purified as described under B) and used for ligation.
• F) The expression vector pNCO113 was digested with the restriction endonucleases EcoRI and BamHI. 25.0 μl pNCO113 [5 μg] 2.5 μl EcoRI (62.5 U) 3.0 μl BamHI [60 U] 24.0 μl OPAU (10 ×) 65.5 μl H ₂ O _bidist. The batch also became Incubated for 180 min at 37 ° C and the resulting DNA fragment with a length of 3387 bp as described under B) purified and used for ligation.
• G) Ligation of the DNA fragments from E) and F) in a molar ratio of 3: 1 (Sgamarella, 1979) 1 μl expression vector from F) [50 fmol] 2 μl DNA fragment from E) [150 fmol] 4 ul H ₂ O _bidest mix, 10 min at 55 ° C and then heat for 5 min on ice provide 2 ul T ₄ buffer (5 x; 250 mM Tris / HCl, pH 7.6; 50 mM MgCl _2; 5 mM ATP; 5 mM DTT; 25% (w / v) polyethylene glycol-8000) 1 μl T ₄ ligase [1 U] (Gibco BRL, Eggenstein) The batch was incubated overnight at 4 ° C to obtain the plasmid pNCO-BS-LuSy.
• H) Preparation of electrocompetent Escherichia coli XL1 cells (Dower et al., 1988): 1 liter of LB medium was inoculated 1: 100 with an overnight culture grown at 28 ° C. and incubated at 37 ° C. in a shaking incubator to an optical density (600 nm) from 0.5 to 0.7 (early to medium logarithmic phase). The bacterial culture was placed on ice for 15 min to stop cell growth and then centrifuged to obtain cell mass (Sorvall GS-3 rotor, 2300 rpm ^-1 , 4 ° C, 15 min). The cell pellet was then suspended in 1 liter of ice cold and sterile 10% glycerol solution and centrifuged again. This was followed by two more washes (500 ml of 10% glycerol solution, 20 ml of 10% glycerol solution) followed by centrifugation. After the last centrifugation step, the residue was taken up in 2-3 ml of 10% glycerol solution and placed on ice. Electroporation cell and cuvette holder were now pre-cooled on ice. In a precooled 0.5 ml Eppendorf tube, 40 μl of the prepared cell suspension were mixed with 1-2 μl of the ligation mixture from G) and then pipetted into the pre-cooled electroporation cell (0.1 cm). Electroporation was carried out at 25 μF, 1.8 kV, 200 Ω (BioRad, Munich), with a time constant of electroporation of 4-5 msec. Immediately after the electroporation, the cell suspension was treated with 1 ml of SOC medium (2% peptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl ₂ , 10 mM MgSO ₄ , 20 mM glucose) transferred sterile 10 ml vessel. For phenotypic expression, the cells were brewed for 1 h at 37 ° C. in a shaker and subsequently incubated in 20 μl and 200 μl portions on LB-Amp plates (21 g / l agar, 10 g / l casein hydrolyzate, 5 g / l yeast extract, 5 g / l NaCl; 150 mg / l ampicillin) and incubated overnight at 37 ° C to obtain the expression strain XL1-pNCO-BS-LuSy.
• I) The isolation of the expression plasmids (pNCO-BS-LuSy) from the expression strains obtained under H) (XL1-pNCO-BS-LuSy) was carried out according to Birnboim and Doly (1979). Cells from a 100 ml overnight culture (10 g / l casein hydrolyzate, 5 g / l yeast extract, 5 g / l NaCl, 150 mg / l ampicillin) were dissolved in 4 ml buffer S1 (50 mM Tris / HCl, 10 mM EDTA, 100 μg Rnase A / ml, pH 8.0) and then admixed with 4 ml of buffer S2 (200 mM NaOH, 1% SDS). After careful swirling of the sample vessel and a 5 minute incubation at room temperature, the mixture was mixed with 4 ml of buffer S3 (2.6 M KAc, pH 5.2), mixed and placed on ice for 20 minutes. After centrifugation (SS34 rotor, 17000 rpm ^-1, 4 ° C, 30 min), the supernatant using Nucleobond AX100 ^® columns (Macherey and Nagel, Duren) was purified. The column material was equilibrated with 2 ml of buffer N2 (0.9 M KCl, 100 mM Tris-phosphate, pH 6.3, 15% (v / v) ethanol) and then the supernatant was applied. After leaching of impurities with 8 ml of buffer N3 (1.3 M KCl, pH 6.3), the DNA was eluted with 2 ml of buffer N5 (1.3 M KCl, pH 8.0). From the eluate, the DNA was precipitated with 0.7 times the volume of isopropanol, washed twice with ice-cold 70% ethanol, dried in a vacuum centrifuge and taken up in 200 μl bidistilled water.
• J) Sequencing of the plasmid DNA (pNCO-BS-LuSy) was performed according to the chain termination method of Sanger et al. (1971) with labeled terminators and Taq DNA polymerase. The sequencing batch contained 1 μg plasmid DNA from I), 10 pmol sequencing oligonucleotide Seq-1 (5 'gtg agc gga taa caa tttacac aca g 3'), 10 μl Terminator Premix ^™ (dNTP's, ddNTP's, labeled ddNTP's and Taq DNA Polymerase) of ABI (Weiterstadt) and was with bidest. Water is made up to a final volume of 21 μl (protected from light since labeled ddNTPs are light-sensitive). The reaction was carried out in a GeneAmpPCR System 2400 machine from Perkin Elmer (Norwalk, Connecticut, USA) with the following program: 15 s / 96 ° C (denaturation of the DNA) 15 s / 50 ° C (annealing of the primer) 4 min / 72 ° C (Propagation of DNA) The polymerase reaction was carried out 25 times in succession. After the polymerase reaction, 80 μl were redistilled. Water was added to the batch and extracted twice with 100 .mu.l of phenol / chloroform / amyl alcohol mixture (25: 24: 1) from ABI (Weiterstadt), wherein the heavier, reddish colored, chloroform-containing phase was discarded. The precipitation of the DNA was carried out with 300 .mu.l of absolute ethanol in the presence of 10 .mu.l of a 3 M sodium acetate solution. The DNA was then washed strength by centrifugation (Eppendorf rotor, 14000 rpm ^-1, RT, 30 min) pelleted, the residue treated with ice-cold 70% ethanol solution and dried in the vacuum centrifuge. The dry DNA pellet was taken up in a mixture of 1 .mu.l of 50 mM EDTA, pH 8.0 and 5 .mu.l of formamide and heated to 95.degree. C. for 2 minutes to denature the DNA and then cooled immediately on ice. 1.5 μl of these samples were then applied to a 4.75% polyacrylamide sequencing gel. To prepare the sequencing gel, National Diagnostics (Atlanta, Georgia, USA) added 13.3 ml of UltraPureSequagel ^™ Sequencing System Concentrate to 49.7 ml UltraPureSequagel ^™ Sequencing System Diluent and deionized with a Amberlite MB-1 spatula tip. The suspension was then filtered (0.2 μm filter) and 7 ml of UltraPureSequagel ^™ Sequencing System buffer added. After degassing under water jet vacuum for 10 minutes, 210 μl of a 10% ammonium peroxodisulfate solution and 25 μl of TEMED were added and poured into a prepared gel carrier. The separation of the labeled DNA fragments was carried out in TBE buffer (1 M Tris base, pH 8.3, 0.85 M boric acid, 10 mM EDTA) over a period of 7 h on a Prism ^™ 377 DNA sequencer of Perkin-Elmer-ABI (Weiterstadt).
• K) expression plasmids containing a correctly incorporated lumazine synthase gene were then cultured in LB-AMP medium (10 g / l casein hydrolyzate, 5 g / l yeast extract, 5 g / l NaCl, 150 mg / l ampicillin). Cultures were performed in a volume of 25 ml in 100 ml Erlenmeyer flasks. An overnight culture (approximately 15 ml) of cells from H), which originated from a single colony, was first inoculated. The main culture was inoculated 1:50 (v / v) with the overnight culture. The growth of liquid cultures was monitored by measuring the optical density at 600 nm (OD ₆₀₀ ) against un-grown medium as the zero value. The expression strains were grown to an OD ₆₀₀ of 0.7 and then the transcription of the lumazine synthase gene was induced with IPTG (isopropyl-β-D-thiogalactopyranoside) at a concentration of 2 mM. After fermentation (after induction for a further 5 h), the cells were harvested by centrifugation (5000 rpm ^-1 4 ° C, 15 min), washed with saline (20% of the main culture volume, saline: 0.9% NaCl soln.) And stored at -20 ° C until further use.
• L) The digestion of the cells from K) was carried out using an ultrasound-generating apparatus (Branson Sonifier B-12A, Branson Sonic Power Company, Dunbury, Connecticut). For digestion, the washed cell pellet was suspended in 800 μl of digestion buffer (50 mM K-phosphate, pH 7.0, 10 mM EDTA, 10 mM Na ₂ SO ₃ , 0.3 mM PMSF, 0.02% sodium azide) and stirred for 10 min chilled on ice. The cell suspension was then treated for 8 sec with the needle probe of the ultrasound machine at level 4.5. Subsequently, the suspension was cooled for 5 min on ice and subjected to another ultrasonic treatment. The cell suspension was then zentrifungiert (Eppendorff centrifuge, 15000 rpm ^-1 4 ° C, 15 min) was used and the supernatant (crude extract) for further processing.
• M) To check the expression rate or the size of the monomeric protein strand (lumazine synthase), a denaturing polyacrylamide electrophoresis according Laemmli (1970) was performed. Routinely, gels were made with 4% acrylamide in the stacking gel and 16% acrylamide in the separating gel (Acrylamide stock solution: 38.8% (w / v) acrylamide; 1.2% (w / v) N, N'-methylenebisacrylamide). The crude extracts from L) were 1: 2; Diluted 1: 5 or 1:10 with protein loading buffer (20% glycerol; 4% 2-mercaptoethanol; 4% (w / v) SDS; 0.05% bromophenol blue) and boiled for 15 min on a boiling water bath. After cooling, the remaining insoluble components of the samples were pelleted in an Eppendorf centrifuge (15,000 rpm ^-1 , 5 mm, 4 ° C.) and 8 μl of the clear supernatant were applied to the collection gel. Dalton Mark VII-L from Sigma (Deisenhofen) with protein bands at 66, 44, 36, 29, 24, 20 and 14 kDa (hereinafter referred to as marker proteins) served as the molecular weight standard. The electrophoresis was performed at a constant 20 mV per SDS gel. The staining of the protein bands was then carried out using Coomassie staining solution (40% methanol, 10% acetic acid, 0.2% (w / v) Coomassie Blue R 250). For decolorization of the non-protein matrix-added acrylamide gel, a Coomassie decolorizing solution was used (40% methanol, 10% acetic acid (tech.), 50% water). In the crude extract of the strain XL1-pNCO-BS-LuSy a protein band with a molecular weight of about 16 kDa could be observed, which was not visible in a control strain without pNCO-BS-LuSy expression plasmid. The observed band corresponded to a share of about 10% based on all soluble proteins (estimated).
• N) The functionality of the 6,7-dimethyl-8-ribityllumazine synthase was checked using the natural substrates (5-amino-6-ribitylamino-2,4 (1H, 3H) -pyrimidinedione and L-3,4-dihydroxy 2-butanone-4-phosphate; Bacher et al., 1997). The mixture contained 100 mM K-phosphate buffer pH 7.0, 4 mM EDTA, 0.6 mM 5-amino-6-ribitylamino-2,4 (1H, 3H) -pyrimidinedione (PYR, prepared by catalytic hydrogenation of 5 Nitro-6-ribitylamino-2,4 (1H, 3H) -pyrimidinedione), 2 mM DTT, 1 mM L-3,4-dihydroxy-2-butanone-4-phosphate (DHP) and crude extract from L). The mixture was first preincubated for 3 min without PYR and the enzyme reaction then started by addition of PYR. The assay was incubated at 37 ° C. Portions were taken from the mixture at different time intervals (2, 5 and 10 minutes), the reaction stopped by the addition of TCA (15% in water) and centrifuged (15000 rpm ^-1 , 5 min, RT). The evaluation was carried out by HPLC on the reverse phase Nucleosil 10C ₁₈ (4 × 250 mm). The concentration of 6,7-dimethyl-8-ribityllumazine formed was determined by fluorescence detection (excitation: 407 nm, emission: 487 nm). The elution buffer contained 7% methanol and 30 mM formic acid. The standard used was chemically synthesized 6,7-dimethyl-8-ribityllumazine. One unit (1 U) of 6,7-dimethyl-8-ribityllumazine synthase catalyses the formation of 1 nmol of 6,7-dimethyl-8-ribityllumazine per hour at 37 ° C. In the crude extract of the strain XL1-pNCO-BS-LuSy a volume activity of about 15600 U / ml could be measured. After determining the total protein concentration to O) (13 mg / ml), a specific activity of about 1200 U / mg could be calculated.
• O) The determination of the protein in the crude extract was carried out according to a variant of the method of Bradford (Read and Northcote, 1981, Compton and Jones, 1985). The dye reagent was recovered by adding 0.1 g of Serva Blue G, 100 ml of 16 M phosphoric acid and 47 ml of ethanol in water, filtered and stored in the dark at 4 ° C. The crude extracts were diluted 50-fold with a buffer containing 2.0 g of Na ₂ HPO ₄ , 0.6 g of KH ₂ PO ₄ , 7.0 g of NaCl and 0.2 g of sodium azide per liter of water. 950 μl of reagent were added to 50 μl of this solution and incubated for 15 min at room temperature. Subsequently, the absorbance at 595 nm was determined against a blank from 50 μl buffer to 950 μl reagent. For each sample, a triple determination was made. The results were converted into protein concentrations by means of a calibration line obtained with BSA (bovine serum albumin) as standard.
• P) For the negative contrast was used as carrier meshes coated with Formvar / coal. These were each before the New use (see standard protocols for performing Negative contrast on the electron microscope). About 10 μl protein solution (≈ 1 mg / ml) were put on the net. The part that does not work after 1 min to the carrier material was adsorbed, was removed. Subsequently, the proteinaceous Layer several times alternately with uranyl acetate solution (2% in water) and bidest. Water wets. The last drop of uranyl acetate was about 30 sec left on the drug. Finally became the carrier mesh dried with filter paper and in the support apparatus of the electron microscope used (transmission electron microscope JEM-100CX, Jeol, Japan). Negative contrasts showed hollow, spherical particles with an outside diameter of about 15 nm and an inner diameter of about 5 nm.
• Q) Western blot analysis was performed by the method of Sambrook et al. (1989). Starting from a denaturing SDS-polyacrylamide gel (16%), the denatured proteins were transferred over a period of 2 hours at a constant current of 40 mA. Following transformation of the proteins onto the membrane, they were washed briefly in Antibody Wash Buffer A (20mM Tris, pH 7.4, 150mM NaCl, 3mM KCl, 0.05% Tween 20) and then washed in Antibody Wash Buffer B (with 3% milk powder) incubated for 1 hour. Thereafter, the membrane was swirled overnight with 10 μl of anti-sRFS (rabbit crude serum with polyclonal antibodies against lumazine synthase, diluted 1:10 in 1 mg / l BSA) in a total volume of 5 ml of Antibody Wash Buffer C (with 1% milk powder) , After incubation, the membrane was washed 3x with 5 ml each of Antibody Wash Buffer-A. The rinsed membrane was then incubated for 1 hour with 20 μl of anti-rabbit IgG-HRP conjugate (in 50% glycerol) in Antibody Wash Buffer-C. After washing three times, the lumazine synthase-containing bands were detected by addition of 3,3'-diaminobenzidine (6 mg in 10 ml of Antibody Wash Buffer A) and 10 μl of hydrogen peroxide (30%). After development of the membrane, the lumazine synthase could be detected at a molecular weight of about 16.2 kDa.
• R) The lumazine synthase from the strain XL1-pNCO-BS-LuSy was purified in two steps: The cultivation of the Escherichia coli cells was carried out according to K), but in a volume of 1 l. For digestion, the washed cell pellet was suspended in 32 ml digestion buffer and cooled on ice for 10 min. The cell suspension was then treated 15 times with the needle probe of the sonicator (Branson Sonifier B-12A, Branson SONIC Power Company, Dunbury, Connecticut) at level 5. Subsequently, the suspension was cooled on ice for 5 min. The process was repeated a total of 4 times. The digested cell suspension was then centrifuged (Sorvall SS34 rotor, 15000 rpm ^-1 , 4 ° C) and the clear supernatant applied to an anion exchange column (DEAE-cellulose DE52, 2 x 15 cm, Whatman Ltd., Maidstone, UK). The column was first rinsed with 100 ml of Buffer A (50 mM K-phosphate, 10 mM EDTA, 10 mM sodium sulfite, 0.02% sodium azide, pH 7.0) and the lumazine synthase subsequently subjected to an elution gradient (101 ml to 200 ml 15% Buffer B; 201 ml to 500 ml 18% Buffer B; 501 ml to 650 ml 100% Buffer B) including Buffer B (1M K-phosphate, 10 mM EDTA, 10 mM sodium sulfite, 0.02% sodium azide, pH 7.0) (flow rate: 1 ml / min). The lumazine synthase could be eluted at about 250 mM K-phosphate. The fractions were then tested for N) for lumazine synthase activity. Active fractions were pooled and dialyzed against buffer A in a 1: 1000 ratio (18h, 4 ° C). The dialyzate was then concentrated using an ultracentrifuge (Beckman LE 70 with 70Ti fixed angle rotor, 32,000 rpm ^-1 , 18h, 4 ° C). The approximately 75% enriched concentrated protein solution was applied to a gel filtration column equilibrated with buffer A (Sepharose-6B, 2 × 180 cm, Pharmacia Biotech, Freiburg) and eluted with buffer A (flow rate: 0.5 ml / min). The lumazine synthase eluted shortly after the exclusion limit. The fractions were then assayed for lumazine synthase activity after N) and then concentrated using an ultracentrifuge (Beckman LE 70 fixed angle rotor 70Ti, 32,000 rpm ^-1 , 18h, 4 ° C). The purity check was carried out according to M) (SDS-PAGE), whereby only one protein band was observed at about 16 kDa. After determination of the enzymatic activity to N) and the protein concentration to O) a specific activity of 12400 U / mg could be calculated. Negative contrasts according to P) showed hollow, spherical particles with an outer diameter of about 15 nm and an inner diameter of about 5 nm.
• S) To check the quaternary structure of the purified protein, native electrophoresis was performed using 3.5% polyacrylamide flats. 5.7 ml Acrylamide stock solution (38.8% (w / v) acrylamide; 1.2% (w / v) N, N'-methylenebisacrylamide), 46 ml gel buffer (0.2 M Na phosphate, pH 7.2 ), 13 ml of _bidistilled H ₂ O, 300 μl of ammonium peroxodisulfate solution (10% (w / v) in water), 65 μl of TEMED (N, N, N ', N'-tetramethylethylenediamine) and 5 mg of bromophenol blue were mixed and placed in a gel casting booth (Pharmacia Biotech, Freiburg) molded using a carrier film (GelBond PAG ^® film, FMC Bioproducts, Rockland, ME, USA) and polymerized overnight at room temperature. On the gel was added 20 μl of protein solution at a concentration of 0.2-1 mg / ml. The gel buffer was used as the electrode buffer. The gel was developed at constant 100 mA under temperature control (10 ° C). Staining or decolorization was carried out as described under M). The purified lumazine synthase was seen as a discrete band on the native gel and showed the same running behavior as a sample purified from wild-type Bacillus subtilis.
• T) To verify the quaternary structure or the assessment of the structural homogeneity of the purified lumazine synthase, a sedimentation analysis was carried out on the analytical ultracentrifuge (Optima XLA with rotor AN60 Ti, Beckman Instruments, Munich) (Laue et al., 1992). To determine the rate of sedimentation the lumazine was centrifuged at 45,000 rpm ^-1. Every 5 min, the radial absorption change was measured at 280 nm and thus the sedimentation course of the protein was monitored. Recombinant lumazine synthase sedimented as a homogeneous, symmetric boundary layer, ie, the sample was a single molecular species. From the temporal sedimentation progress during the individual measurements an average sedimentation constant S _{20, w} of 26.3 S was determined.
• U) The exact determination of the native molecular weight of the lumazine synthase and the final confirmation of the degree of aggregation was carried out with the analytical ultracentrifuge according to the sedimentation equilibrium method. The radius-dependent concentration measurement was carried out by measuring the absorbance at 280 nm. The samples were concentrated protein (according to Sepharose-6B), which showed only a single band (16.2 kDa) on SDS-polyacrylamide gels. The absorbance of the samples used was about 0.3 at 280 nm. 150 μl sample was placed in the sample sector of a measuring cell and underlaid with 15 μl oil (Fluorochemical FC 43, Beckman). The reference sector was filled with 200 μl buffer A (see R)). The enzyme sample was trifugiert zen at 3000 rpm ^-1 and 4 ° C until the Equilibrium weight between centrifugation and back diffusion had set. The evaluation of the data was carried out with the software XLA-Data-Analysis (subprogram Origin-single, Beckman Instruments). The partial specific volume of the protein was calculated approximately according to Cohn and Edsall (1943) from the partial specific volumes of the amino acids and addition of a temperature correction. For the lumazine synthase, a molecular mass of 925 kDa was obtained at 4 ° C, thus confirming aggregation to a 60-mer.

Example 2

Homologous expression of Lumazine synthase from Bacillus subtilis in the Bacillus subtilis strain BR151-pBL1

• A) Analog Beispiel 1 A)–E), wobei anstelle des Oligonukleotids EcoRI-RBS-1 Oligonukleotid EcoRI-RBS-2 (5' ata ata gaa ttc att aaa gag gag aaa tta act atg 3') verwendet wurde.
• B) Der Expressionsvektor p602/-CAT wurde analog Beispiel 1 F) verdaut. Das entstandene DNA-Fragment mit einer Länge von 5269 bp wie unter Beispiel 1 B) beschrieben gereinigt und zur Ligation eingesetzt.
• C) Die Ligation erfolgte analog Beispiel 1 G), wobei das Expressionsplasmid p602-BS-LuSy erhalten wurde.
• D) Die Transformation von Escherichia coli XL1-Zellen erfolgte analog Beispiel 1 H), jedoch wurden anstelle LB-AMP-Platten, LB-KAN-Selektivagarplatten (21 g/l Agar; 10 g/l Caseinhydrolysat; 5 g/l Hefeextrakt; 5 g/l NaCl; 15 mg/l Kanamycin) verwendet, da der Vektor p602/-CAT über eine Kanamycin-Resistenz anstelle der Ampicillin-Resistenz verfügt.
• E) Die Isolierung der Expressionsplasmide erfolgte analog Beispiel 1 I), jedoch wurden die Zellen auf LB-KAN-Flüssigmedium (10 g/l Caseinhydrolysat; 5 g/l Hefeextrakt; 5 g/l NaCl; 15 mg/l Kanamycin) angezogen.
• F) Die Sequenzierung erfolgte analog Beispiel 1 J) jedoch mit Sequenzieroligonukleotid Seq-2 (5' gta taa tag att caa att gtg agc gg 3').
• G) Die Kultivierung der Expressionsstämme erfolgte analog Beispiel 1 K), jedoch unter Verwendung von LB-KAN-Flüssigmedium.
• H) Der Aufschluß der Zellen wurde analog Beispiel 1 L) durchgeführt.
• I) Die SDS-Polyacrylamidgelelektrophorese des Escherichia coli Stammes XL1-p602-BS-LuSy (durchgeführt gemäß Beispiel 1 M)) zeigte eine diskrete Proteinbande bei ca. 16 kDa, die einer Expressionsrate von ca. 30 % bezogen auf die gesamte lösliche Proteinfraktion entsprach.
• J) Die Überprüfung der Funktionalität gemäß Beispiel 1 N) und der Gesamtproteinkonzentration gemäß Beispiel 1 O) ergab eine spezifische Aktivität von ca. 3700 U/mg.
• K) Die Herstellung elektrokompetenter Bacillus subtilis Zellen erfolgte nach einer modifizierten Vorschrift von Brigidi et al. (1989). 500 ml LB-ERY-Flüssigmedium (10 g/l Caseinhydrolysat; 5 g/l Hefeextrakt; 5 g/l NaCl; 15 mg/l Erythromycin) wurden 1:100 mit einer Bacillus subtilis Zellen (BR151[pBL1]) enthaltenden Übernacht-Kultur angeimpft und bei 32 °C bis zu einer optischen Dichte (578 nm) von 0,6 bebrühtet. Zum Abstoppen des Zellwachstums wurden die Zellen 30 min auf Eis gestellt und anschließend zur Gewinnung der Zellmasse zentrifugiert (Sorvall-GSA-Rotor, 2300 Umin^–1, 4 °C, 15 min). Der Überstand wurde verworfen, das Zellpellet mit 300 ml eiskaltem, sterilem 1 mM HEPES-Puffer (1 mM (N-2-hydroxyethylpiperazin-N'-ethansulfonsäure in Wasser, pH 7,0) gewaschen und erneut zentrifugiert. Das Sediment wurde zweimal in je 200 ml PEB-Puffer (272 mM Saccharose; 1 mM MgCl₂; 7 mM K-Phosphat, pH 7,4) gewaschen und zentrifugiert. Das Zellpellet wurde abschließend in 16 ml PEB-Puffer suspendiert und auf Eis gekühlt. Elektroporationszelle und Zellenhalter wurden 15 min auf Eis vorgekühlt. 800 μl Zellsuspension wurde in einem vorgekühlten Eppendorf-Gefäß mit 500–1500 ng Plasmid-DNA (p602-BS-LuSy aus E)) vermischt und 10 min auf Eis inkubiert. Nach Überführung der inkubierten Suspension in die Elektroporationszelle (4 mm) wurde die Elektroporation bei 25 μF und 2,5 kV in einer Elektroporationsapparatur (Gene Pulse, BioRad, München) durchgeführt. Anschließend wurde die Zellsuspension für weitere 10 min auf Eis inkubiert. Zur Expression des Phänotyps (Kanamycin-Resistenz) wurden die transformierten Zellen in 6 ml LB-ERY-Medium (s.o.) pipettiert und 2 h bei 32 °C bebrühtet. Anschließend wurden 25 ml LB-ERY-KAN-Medium (10 g/l Caseinhydrolysat; 5 g/l Hefeextrakt; 5 g/l NaCl; 15 mg/l Erythromycin; 15 mg/ml Kanamycin) mit 1 ml des Transformationsansatzes beimpft und für 4–8 h bei 32 °C im Schüttelinkubator bebrühtet. Parallel dazu wurden Portionen von 100 μl, 200 μl und 400 μl des Transformationsansatzes direkt auf LB-ERY-KAN-Selektivagarplatten (21 g/l Agar; 10 g/l Caseinhydrolysat; 5 g/l Hefeextrakt; 5 g/l NaCl; 15 mg/l Erythromycin; 15 mg/ml Kanamycin) ausplattiert. Portionen der Flüssigkultur wurden nach 2, 4, 6 und 8 h auf LB-ERY-KAN-Platten ausplattiert.
• L) Die erhaltenen Bacillus subtilis Stämme BR151-pBL1-p602-BS-LuSy wurden unter Verwendung der Polymerase-Kettenreaktion auf Anwesenheit des Expressionsplasmides getestet. Zur Analytik der Transformanden wurden jeweils PCR-Ansätze (vgl. Beispiel 1 A) jedoch ohne Matrizen-DNA und entsprechend mehr Wasser) vorgelegt und darin unter Verwendung von sterilen Zahnstochern frisches Zellmaterial (von den erhaltenen Kolonien) suspendiert. Nach erfolgter Suspendierung wurde eine Kopie der jeweiligen Transformande auf einer LB-ERY-KAN-Selektivagarplatte angelegt. Bei allen getesteten Kolonien wurde ein DNA-Fragment mit einer Länge von 498 bp erhalten.
• M) Die Kultivierung der Zellen erfolgte auf LB-ERY-KAN-Flüssigmedium (10 g/l Caseinhydrolysat; 5 g/l Hefeextrakt; 5 g/l NaCl; 15 mg/l Erythromycin; 15 mg/ml Kanamycin) gemäß Beispiel 1 K), jedoch wurde die Fermentation bei 32 °C durchgeführt und die Wachstumsphase nach Induktion mit IPTG betrug 18 h.
• N) Der Aufschluß der Zellen erfolgte analog Beispiel 1 L).
• O) Die Überprüfung der Expressionsrate anhand einer SDS-Polyacrylamidgelelektrophorese erfolgte analog Beispiel 1 M) und ergab eine Expressionsrate von ca. 40–50 % bezogen auf die gesamte lösliche Proteinfraktion.
• P) Die Überprüfung der Funktionalität bzw. der Proteinkonzentration wurde gemäß Beispiel 1 N) und O) durchgeführt. Im Rohextrakt des Expressionsstammes BR151-pBL1-p602-BS-LuSy konnte eine spezifische Aktivität von 4000 U/mg gemessen werden.
• Q) Die Durchführung der Negativkontrastierung erfolgte nach Beispiel 1 P), wobei hohle, sphärische Partikel mit einem Außendurchmesser von ca. 15 nm und einem Innendurchmesser von ca. 5 nm zu beobachten waren.
• R) Die Western-Blot-Analyse wurde nach Beispiel 1 Q) durchgeführt und ergab ein vergleichbares Ergebnis.
• S) Da die Expressionsrate im verwendeten Bacillus subtilis Stamm BR151-pBL1-p602-BS-LuSy deutlich höher lag als im Escherichia coli Stamm XL1-pNCO-BS-LuSy konnte die Reinigung mit einer einzigen Säule (Sepharose-6B, 2 × 180 cm, Pharmacia Biotech, Freiburg) durchgeführt werden. Die Kultivierung der Zellen erfolgte nach M), jedoch in einem Volumen von 1l. Der Aufschluß erfolgte nach Beispiel 1 R) in 32 ml Aufschlußpuffer, jedoch wurden dem Aufschlußpuffer 30 mg Lysozym zugesetzt. Es erfolgte zunächst eine Inkubation der Zellsuspension (37 °C, 60 min) an die sich der Ultraschall-Aufschluß gemäß Beispiel 1 R) anschloß. Die aufgeschlossenen Zellsuspension wurde anschließend zentrifugiert (Sorvall SS34-Rotor; 15000 Umin^–1; 4°C), filtriert (Porenweite 0,22 μm) und der klare Überstand auf eine mit Puffer A (gemäß Beispiel 1 R)) equilibrierte Gelfiltrationssäule auf getragen und mit Puffer A eluiert (Flußrate: 0,5 ml/min). Die Lumazinsynthase wurde vom Säulenmaterial nicht retardiert. Die Fraktionen wurden anschließend nach Beispiel 1 N) auf Lumazinsynthaseaktivität getestet und anschließend unter Verwendung einer Ultrazentrifuge konzentriert (Beckman LE 70 mit Festwinkelrotor 70Ti; 32000 Umin^–1, 18 h, 4 °C). Die Reinheitsüberprüfung erfolgte nach Beispiel 1 M) (SDS-PAGE), wobei nur eine Proteinbande bei ca. 16 kDa zu beobachten war. Nach Bestimmung der enzymatischen Aktivität und der Proteinkonzentration (gemäß Beispiel 1 N) und O)) konnte eine spezifische Aktivität von 12400 U/mg berechnet werden. Negativkontrastierungen (gemäß Beispiel 1 P)) zeigten hohle, sphärische Partikel mit einem Außendurchmesser von ca. 15 nm und einem Innendurchmesser von ca. 5 nm.
• T) Die Überprüfung der Quartärstruktur der gereinigten Lumazinsynthase erfolgte gemäß Beispiel 1 S) bis U) und ergab ein vergleichbares Ergebnis.

In comparison to heterologous expression in Escherichia coli, a higher yield of lumazine synthase can be achieved in homologous expression in Bacillus subtilis.

• A) Analogously to Example 1 A) -E), using instead of the oligonucleotide EcoRI-RBS-1 oligonucleotide EcoRI-RBS-2 (5 'ata ata gaa ttc att aaa gag gag aaa tta act atg 3') was used.
• B) The expression vector p602 / -CAT was digested analogously to Example 1 F). The resulting DNA fragment with a length of 5269 bp as described in Example 1 B) purified and used for ligation.
• C) The ligation was carried out analogously to Example 1 G), whereby the expression plasmid p602-BS-LuSy was obtained.
• D) The transformation of Escherichia coli XL1 cells was carried out analogously to Example 1 H), but instead of LB-AMP plates, LB-KAN selective agar plates (21 g / l agar, 10 g / l casein hydrolyzate, 5 g / l yeast extract; 5 g / l NaCl, 15 mg / l kanamycin), since the vector p602 / -CAT has kanamycin resistance instead of ampicillin resistance.
• E) The isolation of the expression plasmids was carried out analogously to Example 1 I), but the cells were grown on LB-KAN liquid medium (10 g / l casein hydrolyzate, 5 g / l yeast extract, 5 g / l NaCl, 15 mg / l kanamycin).
• F) The sequencing was carried out analogously to Example 1 J) but with Sequenzieroligonukleotid Seq-2 (5 'gta taa tag att caa att gtg agc gg 3').
• G) The culture of the expression strains was carried out analogously to Example 1 K), but using LB-KAN liquid medium.
• H) The digestion of the cells was carried out analogously to Example 1 L).
• I) The SDS-polyacrylamide gel electrophoresis of the Escherichia coli strain XL1-p602-BS-LuSy (carried out according to Example 1 M)) showed a discrete protein band at about 16 kDa, which corresponded to an expression rate of about 30% based on the total soluble protein fraction ,
• J) The checking of the functionality according to Example 1 N) and the total protein concentration according to Example 1 O) gave a specific activity of about 3700 U / mg.
• K) The preparation of electrocompetent Bacillus subtilis cells was carried out according to a modified protocol of Brigidi et al. (1989). 500 ml of LB-ERY liquid medium (10 g / l casein hydrolyzate, 5 g / l yeast extract, 5 g / l NaCl, 15 mg / l erythromycin) were incubated 1: 100 overnight with Bacillus subtilis cells (BR151 [pBL1]). Seeded culture and brewed at 32 ° C to an optical density (578 nm) of 0.6. To stop cell growth, the cells were placed on ice for 30 min and then centrifuged to obtain cell mass (Sorvall GSA rotor, 2300 rpm ^-1 , 4 ° C, 15 min). The supernatant was discarded, the cell pellet washed with 300 ml of ice-cold, sterile 1 mM HEPES buffer (1 mM N-2-hydroxyethylpiperazine-N'-ethanesulfonic acid in water, pH 7.0) and recentrifuged 200 ml of PEB buffer (272 mM sucrose, 1 mM MgCl ₂ , 7 mM K-phosphate, pH 7.4) were washed and centrifuged, and the cell pellet was finally suspended in 16 ml PEB buffer and cooled on ice, electroporation cell and cell holder were pre-cooled on ice for 15 min, 800 μl of cell suspension was mixed in a pre-cooled Eppendorf tube with 500-1500 ng of plasmid DNA (p602-BS-LuSy from E)) and incubated on ice for 10 min. After transfer of the incubated suspension into the electroporation cell (4 mm), the electroporation was carried out at 25 μF and 2.5 kV in an electroporation apparatus (Gene Pulse, BioRad, Munich). Subsequently, the cell suspension was incubated for a further 10 min on ice. To express the phenotype (kanamycin resistance), the transformed cells were pipetted into 6 ml LB-ERY medium (see above) and brewed for 2 h at 32 ° C. Subsequently, 25 ml of LB-ERY-KAN medium (10 g / l casein hydrolyzate, 5 g / l yeast extract, 5 g / l NaCl, 15 mg / l erythromycin, 15 mg / ml kanamycin) were inoculated with 1 ml of the transformation mixture and Brewed for 4-8 h at 32 ° C in a shaking incubator. In parallel, portions of 100 μl, 200 μl and 400 μl of the transformation mixture were applied directly to LB-ERY-KAN selective agar plates (21 g / l agar; 10 g / l casein hydrolyzate; 5 g / l yeast extract; 5 g / l NaCl; mg / l erythromycin; 15 mg / ml kanamycin). Portions of the liquid culture were plated out after 2, 4, 6 and 8 hours on LB-ERY-KAN plates.
• L) The obtained Bacillus subtilis strains BR151-pBL1-p602-BS-LuSy were tested for the presence of the expression plasmid using the polymerase chain reaction. To the analysis of the Transfor In each case, PCR mixtures (compare Example 1 A) but without template DNA and correspondingly more water) were initially introduced and suspended therein fresh cell material (from the colonies obtained) using sterile toothpicks. After suspension, a copy of each transformant was applied to an LB-ERY-KAN selective agar plate. For all colonies tested, a 498 bp DNA fragment was obtained.
• M) The cells were cultivated on LB-ERY-KAN liquid medium (10 g / l casein hydrolyzate, 5 g / l yeast extract, 5 g / l NaCl, 15 mg / l erythromycin, 15 mg / ml kanamycin) according to Example 1 K ), however, the fermentation was carried out at 32 ° C and the growth phase after induction with IPTG was 18 h.
• N) The digestion of the cells was carried out analogously to Example 1 L).
• O) The expression rate was checked by SDS-polyacrylamide gel electrophoresis analogously to Example 1 M) and gave an expression rate of about 40-50% based on the total soluble protein fraction.
• P) The check of the functionality or the protein concentration was carried out according to Example 1 N) and O). In the crude extract of the expression strain BR151-pBL1-p602-BS-LuSy a specific activity of 4000 U / mg could be measured.
• Q) The negative contrasting was carried out according to Example 1 P), wherein hollow, spherical particles having an outer diameter of about 15 nm and an inner diameter of about 5 nm were observed.
• R) The Western blot analysis was carried out according to Example 1 Q) and gave a comparable result.
• S) Since the expression rate in the Bacillus subtilis strain BR151-pBL1-p602-BS-LuSy used was significantly higher than in the Escherichia coli strain XL1-pNCO-BS-LuSy, purification could be carried out with a single column (Sepharose-6B, 2 × 180 cm , Pharmacia Biotech, Freiburg). The cultivation of the cells took place according to M), but in a volume of 1 l. The digestion was carried out according to Example 1 R) in 32 ml digestion buffer, but 30 mg of lysozyme were added to the digestion buffer. There was first an incubation of the cell suspension (37 ° C, 60 min) to which the ultrasound digestion according to Example 1 R) followed. The digested cell suspension was then centrifuged (Sorvall SS34 rotor, 15000 rpm ^-1 , 4 ° C), filtered (pore size 0.22 μm), and the clear supernatant was supported on a gel filtration column equilibrated with Buffer A (according to Example 1 R)) and eluted with buffer A (flow rate: 0.5 ml / min). The lumazine synthase was not retarded by the column material. The fractions were then tested for lumazine synthase activity according to Example 1 N) and then concentrated using an ultracentrifuge (Beckman LE 70 with fixed angle rotor 70Ti, 32000 rpm ^-1 , 18 h, 4 ° C). The purity check was carried out according to Example 1 M) (SDS-PAGE), with only one protein band being observed at approximately 16 kDa. After determination of the enzymatic activity and the protein concentration (according to Example 1 N) and O)), a specific activity of 12400 U / mg could be calculated. Negative contrasts (according to Example 1 P)) showed hollow, spherical particles having an outer diameter of about 15 nm and an inner diameter of about 5 nm.
• T) The review of the quaternary structure of the purified lumazine synthase was carried out according to Example 1 S) to U) and gave a comparable result.

Example 3

Replacement of the amino acid cysteine at positions 93 through the amino acid serine in lumazine synthase from Bacillus subtilis

• A) The gene for the lumazine synthase from Bacillus subtilis was cloned with the oligonucleotides PNCO-M2 (5 'aga tatttt cat taa aga gga gaa 3'), which binds at the 3 'end to a part of the ribosomal binding site and at the 5' End contains a non-binding sequence section for deletion of the vectorial EcoRI site and RibH-3 (5 'did tat gga tcc tta ttc aaa tga gcg gtt taa att tg 3') amplified from the plasmid pNCO-BS-LuSy (Example 1). 10 μl PCR Buffer 6 μl Mg ²⁺ [1.5 mM] 8 μl dNTP's [200 μM each] 1 μl PNCO-M2 [0.5 μM] 1 μl RibH-3 [0.5 μM] 1 μl pNCO- BS-lusy [10 ng) 1 ul GoldStar Taq polymerase [0.5 U) (Eurogentec, Seraing, Belgium) 72 ul H ₂ O _bidest The batch was in the thermocycler (GeneAmp ^® PCR system 2400; Perkin Elmer) with the following Conditions performed: 1. 5.0 min 95 ° C 2. 0.5 min 94 ° C 3. 0.5 min 50 ° C 4. 0.5 min 72 ° C 5. 7.0 min 72 ° C 6. Cool to 4 ° C Steps 2.-4. were repeated 20 times.
• B) The PCR approach was as in Example 1 B) to give a 505 bp fragment.
• C) A part of the gene for the lumazine synthase from Bacillus subtilis was with the oligonucleotides PNCO-M1 (5 'gtg agc gga taa caa ttt cac aca g 3'), which in the range the EcoRI site in the expression vector binds and this recognition sequence unaffected lets and C93S (5 'gca gct tca ttc gaa aca taa tcg taa tg 3 '), which the mutation and the interface BstBI (amino acid conservative) to detect the mutation, from the plasmid pNCO-BS-LuSy (Example 1) amplified. The implementation of PCR and purification of the fragment (256 bp) was carried out analogously to A) and B).
• D) Extension of the DNA fragment (256 bp) from C) by combination with DNA fragment (505 bp, representing the entire gene length) from B) and carrying out a PCR. In this PCR approach, the PCR fragments used from B) and C) themselves serve as primers for the polymerase reaction. To achieve an efficient extension both fragments were used in equimolar amounts (500 fmol each). 10 μl buffer 6 μl Mg ²⁺ [1.5 mM] 8 μl dNTP's [200 μM each] 1 μl DNA fragment from B) (500 fmol) 1 μl DNA fragment from C) (500 fmol) 1 μl gold star Taq polymerase [0.5 U] 73 ul H ₂ O _bidest The batch was in the thermocycler (GeneAmp ^® PCR system 2400; Perkin Elmer) under the following conditions: 1. 5.0 min 95 ° C 2 min 0.5 94 ° C 3. 0.5 min 65 ° C 4. 0.5 min 72 ° C 5. 7.0 min 72 ° C 6. Cool to 4 ° C Steps 2.-4. were repeated 20 times.
• E) An unpurified portion of the PCR batch from D) was used as a DNA template for a PCR approach with the primer combination PNCO-M1 / RibH-3. The PCR approach was carried out according to A), wherein however, the PCR steps are repeated 25 times were.
• F) The PCR approach was as in Example 1 B) to give a 528 bp fragment.
• G) Further processing was carried out according to Example 1 E) to I). The introduced Mutation was performed by digesting the plasmid pNCO-BS-LuSy-C93S with the Restriction endonuclease BstBI (TT * CGAA) diagnosed (fragments with 3698 bp and 181 bp).
• H) The sequencing was carried out according to Example 1 J).
• I) Proteinchemical work was carried out according to Example 1 K) to S), wherein no significant difference to the wild-type lumazine synthase recognizable has been.

Example 4

Replacement of the amino acid cysteine at positions 139 through the amino acid serine in lumazine synthase from Bacillus subtilis

• A) The gene for lumazine synthase from Bacillus subtilis was analogous to Example 3 A) with the oligonucleotides PNCO-M2 and RibH-3 amplified from the plasmid pNCO-BS-LuSy (Example 1) and cleaned.
• B) Part of the gene for the lumazine synthase from Bacillus subtilis was analogous to Example 3 B) with the oligonucleotides PNCO-M1 and C139S (5 'ggc aga aac agc tga atc tac acc ttt gtt g 3 '), which the mutation and the interface PvuII (amino acid conservative) to detect the mutation, from the plasmid pNCO-BS-LuSy (Example 1) amplified. The implementation of PCR and purification of the fragment (394 bp) was carried out analogously to Example 3 A) and B).
• C) The further molecular biological procedure corresponded to Example 3 D) to H). The introduced mutation was confirmed by digesting the plasmid pNCO-BS-LuSy-C139S with the restriction endonuclease PvuII (CAG * CTG) (3539 bp and 340 bp fragments).
• D) The further biochemical procedure corresponded to Example 1 K) to S) with no significant difference to wild-type lumazine synthase became recognizable.

Example 5

Replacement of the amino acid cysteine at positions 93 and 139 by the amino acid serine in lumazine synthase from Bacillus subtilis

• A) The preparation of the double mutant pNCO-BS-LuSy-C93 / 139S was carried out analogously to Example 4, but was as DNA template the Plasmid pNCO-BS-LuSy-C93S used.
• B) After biochemical characterization (according to example 1 K) to S)) could no significant difference to the wild-type enzyme be determined.

Construction of expression vectors for N-terminal and C-terminal fusion of foreign proteins to lumazine synthase from Bacillus subtilis

The The following examples describe the production of Escherichia coli expression vectors for fusion of genes or synthetically produced DNA fragments at the 5 'end or to the 3'-end of the gene for the lumazine synthase from Bacillus subtilis. The vectors contain the preferred according to the invention Vector elements, such as promoter from bacteriophage T5, a lac operator sequence, an ampicillin resistance marker and an Escherichia coli compatible one Origin of replication.

Example 6

Vector for the fusion of Foreign DNA at the 5 'end of the gene for the lumazine synthase from Bacillus subtilis

• A) The gene for lumazine synthase from Bacillus subtilis was probed with the oligonucleotides N1 (5'act atg gcg gcg gcg cgt agc tgc gcg gcc gct atg atat ata caa gga aat tta g 3 '), which directly implements a NotI interface before the start codon was generated and RibH-4 (3 'did did gga tcc aaa tta ttc aaa tga gcg gtt taa att tg 3 ') from the plasmid pRF2 (see Example 1) amplified. The PCR was analogously to Example 1 A).
• B) The purification was carried out analogously to Example 1 B), wherein a DNA fragment was obtained with 513 bp.
• C) 10 ng of the isolated DNA from B) subsequently served as Matrix for a second PCR with the oligonucleotides N2 (5 'ata ata gata gaa ttc aaa gag gag aaa tta act atg gcg gcg gcg cgt agc tgc 3 '), which in the 5' direction is a ribosomal binding site and a recognition sequence for introduces the endonuclease EcoRI, and RibH-4.
• D) The further procedure was carried out analogously to Example 1 B), E) to J). The plasmid pNCO-N-BS-LuSy is obtained.

Example 7

Vector for the fusion of Foreign DNA to the 3 'end of the gene for the lumazine synthase from Bacillus subtilis

• A) The gene for lumazine synthase from Bacillus subtilis was amplified with the oligonucleotides EcoRI-RBS-2 (see Example 2 A)) and C2 (5 'ttt tcg gga tcc ttt taa act gtt tgc ggc cgc taa ttc aaa tga gcg gtt taa att tg 3 '), which a NotI interface behind the last coding base triplet of the gene for the lumazine synthase and in the 3 'direction introduces a BamHI site spaced 13 nucleotides apart the plasmid pNCO-BS-LuSy (see Example 1) amplified. The former stop codon of the lumazine synthase gene becomes through that for the amino acid Leucine coding base triplet TTA replaced.
• B) The further procedure was carried out analogously to Example 1 B), E) to J). The plasmid pNCO-C-BS-LuSy is obtained.

Fusion of complete proteins to the N-terminus or to the C-terminus of the lumazine synthase Bacillus subtilis

The The following examples describe the fusion of an intact complete gene to the 5 'end or to the 3 'end of the gene for the Lumazine synthase from Bacillus subtilis. The examples illustrate the feasibility the fusion of complete, biologically active proteins to the N-terminus as well as to the C-terminus the icosahedral lumazine synthase from Bacillus subtilis.

Example 8

Fusion of dihydrofolate reductase from Escherichia coli to the N-terminus of lumazine synthase from Bacillus subtilis

• A) The gene for dihydrofolate reductase (DHFR) was isolated by PCR from isolated chromosomal DNA (Escherichia coli RR28) using the oligonucleotides EC-DHFR-1 (5 'gag gag aaa tta act atg atc agt ctg att gcg g 3 '), which at the 3'-end to the 5'-end of the Complementary to DHFR gene is and at the 5'-end for one Part of an optimized ribosomal binding site encoded, and EC-DHFR-2 (5 'cta gcc gta aat tct ata gcg gcc gca cgc cgc tcc aga atc 3 '), which is complementary to the gene of DHFR at the 3' end and immediately behind the last coding base triplet a recognition sequence for the Introducing endonuclease NotI, amplified. The implementation The PCR was carried out analogously to Example 1 A), wherein about 50 ng chromosomal DNA was used.
• B) The purification of the PCR approach was carried out according to Example 1 B) to give a DNA fragment of 513 bp in length.
• C) The implementation a second PCR was carried out analogously to Example 1 C), but the Oligonucleotide combination BS-MfeI (5 'ata ata caa ttg att aaa gag gag aaa tta act atg 3 '), which completes the ribosomal binding site and immediately before ribosomal binding site a recognition sequence for the restriction endonuclease MfeI into the DNA fragment introduces, and EC-DHFR-2 was used.
• D) The PCR approach was as described in Example 1 B) to give a fragment having a length of 531 bp.
• E) The isolated DNA fragment was digested with the restriction endonucleases MfeI. 30.0 μl Fragment from the 2nd PCR 5.0 μl MfeI (50 U) 10.0 μl Buffer 4 (10 ×, 50 mM K-acetate, 20 mM Tris-acetate, 10 mM Mg-acetate, 1 mM dithiothreitol , pH 7.9) 55.0 _ul H ₂ O _bidist The restriction enzyme was obtained from New England Biolabs (Schwalbach) The batch was incubated at 37 ° C. for 150 min and purified as described under Example 1 B) and for digestion used with the restriction endonuclease NotI.
• F) The purified DNA fragment from E) was digested with the restriction endonuclease NotI. 30.0 μl fragment from E) 5.0 μl NotI [50 U] 10.0 μl Buffer 3 (10 × 100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl ₂ , 1 mM dithiothreitol, pH 7.9 ) 55.0 μl H ₂ O _bidist. The restriction enzyme was obtained from New England Biolabs (Schwalbach). The mixture was incubated at 37 ° C. for 150 minutes and purified as described under Example 1 B) and used for ligation.
• G) 5 μg of the expression vector pNCO-N-BS-LuSy in a volume of 30 μl first digested with the restriction endonuclease NotI analog F) and according to Example 1 B).
• H) The vector fragment from G) was subsequently digested with the restriction endonuclease EcoRI. 30.0 μl vector fragment from G 2.5 μl EcoRI [62.5 U] 20.0 μl OPAU (10 × 500 mM K-acetate, 100 mM Mg-acetate, 100 mM Tris-acetate pH 7.5 47.5 μl H ₂ O _bidist. The batch was likewise incubated at 37 ° C. for 150 min and the resulting 3863 bp DNA fragment was purified as described under Example 1 B) and used for ligation.
• I) The further procedure was carried out according to Example 1 G) to L).
• J) SDS-polyacrylamide gel electrophoresis was carried out according to the example 1 M). In the crude extract of the strain XL1-pNCO-EC-DHFR-BS-LuSy a protein band could be detected be observed with a molecular weight of about 34.5 kDa, the in a control strain without pNCO-EC-DHFR-BS-LuSy expression plasmid not to be seen. The observed gang corresponded to a share from about 40-50 % based on all soluble Proteins (estimated).
• K) The review of functionality 6,7-dimethyl-8-ribityllumazine synthase was carried out according to the example 1 N) in conjunction with O). In the crude extract could no significant Difference to a non-derivatized wild-type lumazine synthase to be watched.
• L) The purification was done according to Example 2 S) on a Sepharose 6B gel filtration column, wherein the concentration of the protein fractions was carried out at 28000 rpm ^-1. Negative contrast according to Example 1 P) showed hollow, spherical particles having an outer diameter of about 20 nm and an inner diameter of about 5 nm.
• M) The quaternary structure was examined according to Example 1 S). Compared to wild-type lumazine synthase, a marked difference in migration rate (EC-DHFR-BS-LuSy wan It was much slower, but also a discrete band.

Example 9

Fusion of the 'maltose-binding protein' from Escherichia coli to the N-terminus of lumazine synthase from Bacillus subtilis

• A) The gene for the "maltose-binding protein" (MBP) was determined by means of PCR from the plasmid pMAL-C2 (New England Biolabs, Schwalbach) under Use of oligonucleotides MALE-1 (5 'gag gag aaa tta act atg aaa atc gaa gaa ggt aaa c 3 '), which at the 3'-end to the 5'-end of the Complementary to MBP gene is and at the 5'-end for one Part of an optimized ribosomal binding site, and MALE-2 (5 'gca ggt cga ctc tag cgg ccg cga att ctg 3 '), which at the 3'-end complementary to the MBP gene is and immediately after the last coding base triplet a recognition sequence for introduces the endonuclease NotI, amplified. The implementation the PCR was carried out analogously to Example 1 A), wherein about 10 ng plasmid DNA was used.
• B) The purification of the PCR approach was carried out according to Example 1 B) to obtain a DNA fragment of 1210 bp in length.
• C) The implementation a second PCR was carried out analogously to Example 1 C), but the Oligonucleotide combination BS-MfeI (5 'ata ata caa ttg att aaa gag gag aaa tta act atg 3 '), which completes the ribosomal binding site and immediately before ribosomal binding site a recognition sequence for the restriction endonuclease Mfel in the DNA fragment introduces, and MALE-2 was used.
• D) The PCR approach was as described in Example 1 B) to give a fragment of 1227 bp in length.
• E) The further procedure was carried out according to Example 8 E) to I).
• F) SDS-polyacrylamide gel electrophoresis was performed according to Example 1 M). In the crude extract of the strain XL1-pNCO-EC-MBP-BS-LuSy a protein band could be detected are observed with a molecular weight of about 59.5 kDa, the in a control strain without pNCO-EC-MBP-BS-LuSy expression plasmid not to be seen. The observed gang corresponded to a share of about 50% based on all soluble Proteins (estimated).
• G) The review of the functionality 6,7-dimethyl-8-ribityllumazine synthase was carried out according to the example 1 N) in conjunction with O). In the crude extract could no significant Difference to a non-derivatized wild-type lumazine synthase to be watched.
• H) The purification was carried out according to Example 2 S) on a Sepharose 6B gel filtration column, wherein the concentration of the protein fractions at 28000 Umin ^{-1 was} performed. Negative contrasting according to Example 1 P) showed hollow, spherical particles having an outer diameter of about 25 nm and an inner diameter of about 5 nm.
• I) The review of quaternary structure took place according to example 1 S). Compared to BS-LuSy and EC-DHFR-BS-LuSy showed a marked difference in migration rate (EC-MBP-BS-LuSy migrated significantly slower than BS-LuSy and EC-DHFR-BS-LuSy, but also formed one discrete band).

Example 10

Fusion of dihydrofolate reductase from Escherichia coli to the C-terminus of lumazine synthase from Bacillus subtilis

• A) The gene for dihydrofolate reductase (DHFR) was isolated by PCR from isolated chromosomal DNA (Escherichia coli RR28) using the oligonucleotide oligonucleotides EC-FolA-1 (5 'ata gtg gcg aca atg cgg ccg ctg gtg gag gcg gaa tga tca gtc tga ttg cgg cg 3 '), which at the 3 'end to the 5' end of the DHFR gene complementary is and at the 5'-end immediately before the start codon of the DHFR gene, a recognition sequence for the Restriction endonuclease Introduces NotI and EC-FolA-2 (5 'ttc gga tcc tta ccg ccg ctc cag aat c 3 '), which at the 3'-end complementary to the gene of DHFR is and immediately after the stop codon a recognition sequence for the Introduces endonuclease BamHI, amplified. The implementation of PCR was carried out analogously to Example 1 A), with about 50 ng chromosomal DNA was used.
• B) The purification of the PCR approach was carried out according to Example 1 B) to give a DNA fragment 524 bp in length.
• C) The isolated DNA fragment was digested with the restriction endonucleases BamHI. 30.0 μl fragment from the 2.PCR 3.0 μl BamHI [60 U] 20.0 μl OPAU (10 ×) 47.0 μl H ₂ O _bidist. The restriction enzyme was obtained from Pharmacia Biotech (Freiburg). The batch was incubated at 37 ° C. for 150 minutes and purified as described under Example 1 B) and used for digestion with the restriction endonuclease NotI.
• D) The purified DNA fragment from C) was digested with the restriction endonuclease NotI according to example 8 F) digested and purified as described in Example 1 B) and used for ligation.
• E) 5 μg of the expression vector pNCO-C-BS-LuSy in a volume of 30 μl first according to C) and D) digested, resulting in a fragment with a length of 3880 bp is, according to example 1 B), and used for ligation.
• I) The further procedure was carried out according to Example 1 G) to L).
• J) SDS-polyacrylamide gel electrophoresis was carried out according to the example 1 M). In the crude extract of the strain XL1-pNCO-BS-LuSy-EC-DHFR, a protein band could be observed with a molecular weight of about 34.8 kDa, the in a control strain without pNCO-BS-LuSy-EC-DHFR expression plasmid not to be seen. The observed gang corresponded to a share of about 25% based on all soluble Proteins (estimated).
• K) The review of functionality 6,7-dimethyl-8-ribityllumazine synthase was carried out according to the example 1 N) in conjunction with O). In the crude extract could no significant Difference to a non-derivatized wild-type lumazine synthase to be watched.
• L) The review of the quaternary structure took place according to example 1 S). Compared to wild-type lumazine synthase could be a clear Difference in migration speed (EC-DHFR-BS-LuSy migrated significantly slower, but also formed a discrete band).

Coupling of a 17 amino acid long Epitops of the VP2 surface protein a mammalian virus to the N-terminus, to the C-terminus, and to both terms of lumazine synthase from Bacillus subtilis

The The following examples describe the fusion of short peptides both terms of the icosahedral lumazine synthase from Bacillus subtilis while preserving the spherical Structure. By the simultaneous assignment of both terms becomes one 120-fold occupancy of the surface of the icosahedron.

The 17 amino acids long peptide counts to the highly conserved areas of the VP2 surface protein of various animal virus species, e.g. Mink enteritis virus, Feline panleukopeniavirus or Canine parvovirus.

Example 11

Fusion of the VP2 domain to the N-terminus of lumazine synthase

• A) The gene for lumazine synthase from Bacillus subtilis was first with the oligonucleotides N-VP2-1 (5'gt cag ccg gct gtt cgt aac gaa cgt atg aat atc ata caa gga aat tta gtt ggt ac 3 '), which at its 3' end is complementary to the 5 'end of the lumazine synthase gene and at the 3'-end for one Part of the VP2 domain coded and RibH-3 (see Example 3 A)) from the plasmid pRF2 (see Example 1 A)) amplified. The PCR was carried out according to the example 1 A).
• B) The PCR approach was as in Example 1 B) to give a DNA fragment of 504 bp in length, purified and as a DNA template for used a second PCR.
• C) The implementation the second PCR was carried out analogously to Example 1 C), except that the oligonucleotide combination N-VP2-2 (5 'gag gag aaa tta act atg ggg gac ggt gct cag ccg gac ggt ggt cag ccg gct gtt cgt aac gaa cg 3 '), which at its 3'-end complementary to the already introduced Part of the VP2 domain is the VP2 domain completed an ATG start codon and a portion of an optimized ribosomal binding site immediately before the VP2 sequence, and RibH-3 were used.
• D) The PCR approach from C) was analogous to Example 1 B), wherein a DNA fragment with a length of 549 bp, purified and used as DNA template for a 3. PCR used.
• E) The implementation the 3rd PCR was carried out analogously to Example 1 C), but with the oligonucleotides EcoRI-RBS-2 (see Example 2 A)) and RibH-3.
• F) The PCR batch from E) was purified analogously to Example 1 B) and a DNA fragment of a length obtained from 567 bp.
• G) Further processing was carried out analogously to Example 1 E) to L), wherein the Escherichia coli expression strain XL1-pNCO-N-VP2-BS-LuSy was obtained.
• H) The review of the expression rate or the size of the monomeric protein strand was carried out analogously to Example 1 M). In the crude extract of the strain XL1-pNCO-N-VP2-BS-LuSy, a protein band with a molecular weight of about 18.2 kDa was observed, which in a comparison strain was not seen without pNCO-N-VP2-BS-LuSy expression plasmid. The observed band corresponded to a share of about 10% based on all soluble proteins (estimated).
• I) After checking the functionality according to example 1 N) to Q) could not be significantly different from the wild-type enzyme to be watched.

Example 12

Fusion of the VP2 domain to the C-terminus of lumazine synthase

• A) The gene for lumazine synthase from Bacillus subtilis was first with the oligonucleotides RibH-1 (see Example 1 A)) and C-VP2-1 (5 'cca ccg tcc ggc tga aca gca ccg tca cct tcg aaa gaa cgg tttag aag ttt gcc 3 '), which on his 3 'end complementary to the gene for the Lumazine synthase is coding immediately after the last one Base triplet of lumazine synthase introduces part of the VP2 domain the plasmid pRF2 (see Example 1 A)) amplified.
• B) The PCR approach was as in Example 1 B) to give a DNA fragment of 506 bp in length, purified and as a DNA template for used a second PCR.
• C) The implementation the second PCR was carried out analogously to Example 1 C), except that the oligonucleotide combination EcoRI-RBS-2 (see Example 2 A)) and C-VP2-2 (5 'ata did gga tcc taa cgt tcg tta cga aca gcc ggc tga cca ccg tcc ggc tga aca gca ccg tc 3 '), which completes the VP2 domain in the 3' direction, behind the last coding base triplet of the VP2 domain is a stop codon into the sequence and immediately after this stop codon, a restriction endonuclease recognition sequence BamHI involves, was used.
• D) The PCR batch from C) was purified analogously to Example 1 B) and a DNA fragment of length 564 bp received.
• E) Further processing was carried out analogously to Example 1 E) to L), wherein the Escherichia coli expression strain XL1 pNCO-C-VP2-BS-LuSy was obtained.
• F) The review of the Expression rate or the size of the monomer Protein strand was carried out analogously to Example 1 M). in the Crude extract of the strain XL1-pNCO-C-VP2-BS-LuSy could produce a protein band be observed with a molecular weight of about 18.1 kDa, the was not seen in a control strain without pNCO-C-VP2-BS-LuSy expression plasmid. The observed band corresponded to a share of about 10% on all soluble Proteins (estimated).
• G) After checking the functionality according to example 1 N) to Q) could not be significantly different from the wild-type enzyme to be watched.

Example 13

Fusion of the VP2 domain to the N- and to the C-terminus of lumazine synthase

• A) Starting from that obtained in Example 11 Expression plasmid pNCO-N-VP2-BS-LuSy expressing the VP2 domain to the 5 'end of the lumazine synthase gene contains fused As in Example 12, the VP2 domain was fused to the 3 'end of the lumazine synthase gene.
• B) The review of the Expression rate or the size of the monomer Protein strand was carried out analogously to Example 1 M). in the Crude extract of strain XL1-pNCO-N / C-VP2-BS-LuSy could produce a protein band be observed with a molecular weight of about 20 kDa, the in a control strain without pNCO-N / C-VP2-BS-LuSy expression plasmid not to be seen. The observed gang corresponded to a share of about 10% based on all soluble Proteins (estimated).
• C) After checking the functionality according to example 1 N) to Q) could not be significantly different from the wild-type enzyme to be watched.

Example 14

Coupling of an artificial 13 amino acids long, in vivo biotinylatable Peptides, to the C-terminus of lumazine synthase from Bacillus subtilis

• A) The gene for the Bacillus subtilis lumazine synthase was amplified using the polymerase chain reaction and the synthetic oligonucleotides EcoRI-RBS-2 (see Example 2 A)) and C-Biotag-1 (5'cat agc ttcga gat gcc gcc gag tgc ggc cgc ttc gaa aga acg gtt taa gtt tgc cat ttc 3 '), which at its 3' end is complementary to the gene for lumazine synthase and immediately after the last coding base triplet of lumazine synthase, DNA for a linker consisting of 3 alanine residues and a Part of the DNA encoding the biotinylatable peptide introduces from the plasmid pNCO-BS-LuSy (see Example 1) on plifiziert. The PCR was carried out analogously to Example 1 A).
• B) The PCR approach was as in Example 1 B) to give a 528 bp DNA fragment, purified and as a DNA template for used a second PCR.
• C) The implementation the second PCR was carried out analogously to Example 1 C), except that the oligonucleotide combination EcoRI-RBS-2 and C Biotag-2 (5 'tat did gga tcc tta gcg cca ctc cat ctt cat agc ttc gaa gat gcc gcc gag tgc ggc 3 '), which is the biotinylatable domain completed in the 3 'direction, immediately behind the last coding base triplet of this domain a stop codon into the sequence and immediately after this stop codon, a restriction endonuclease recognition sequence BamHI involves, was used.
• D) The PCR batch from C) was purified analogously to Example 1 B) and a DNA fragment of length 558 bp received.
• E) Further processing was carried out analogously to Example 1 E) to L), the Escherichia coli expression strain being XL1-pNCO-C biotag-BS-LuSy was obtained.
• F) When reviewing the functionality the lumazine synthase according to Example 1 N) could only have one enzymatic activity, that of the activity of Escherichia coli strain XL1.
• G) The review of the Expression rate or the size of the soluble Proteins were carried out analogously to Example 1 M). In the raw extract of the strain XL1-pNCO-C Biotag-BS-LuSy failed to show any significant protein band are observed with a molecular weight of about 18.5 kDa, the in a control strain without pNCO-C biotag BS-LuSy expression plasmid not to be seen.
• H) To produce a total protein extract, the cells were cultured analogously to Example 1 K). 1/12 of the cell mass obtained was suspended with 300 μl of protein application buffer (see Example 1 M)) and boiled for 15 min on a boiling water bath. After cooling the remaining insolubles of the samples were in an Eppendorf centrifuge pelleted (15000 rpm ^-1, 5 min, 4 ° C) and 8 ul of the clear supernatant to the stacking gel applied. The further procedure was carried out analogously to Example 1 M). It was found in connection with G) that the strain XL1-pNCO-C-Biotag-BS-LuSy recombinant protein in the form of insoluble inclusion bodies in an amount of about 15% based on all cellular proteins (soluble and insoluble proteins, estimated from SDS-PAGE).
• I) To prove the proportion of lumazine synthase in the artificial Lumazine synthase fusion protein was analogous to Western blot analysis Example 1 Q), but in addition to the soluble Protein fraction also the total protein fraction was analyzed, carried out. After development of the membrane could recombinant lumazine synthase fusion protein only in the total protein fraction, but only to a negligible Share in the soluble Protein fraction can be detected.
• J) For detection of biotinylation, both the soluble protein fraction and the total protein fraction were transferred to a membrane. Starting from a denaturing SDS-polyacrylamide gel (16%), the denatured proteins were transferred over a period of 2 hours at a constant current of 40 mA. After the proteins had been transformed onto the membrane, it was washed briefly in antibody wash buffer A (see Example 1 Q)) and then incubated in antibody wash buffer B for 1 hour. Thereafter, the membrane was swirled overnight with 20 μl of streptavidin coupled to 'alkaline phosphatase' (Promega, Madison, Wisconsin, USA) in a total volume of 15 ml of Antibody Wash Buffer-C. After incubation, the membrane was washed 3x with 5 ml each of Antibody Wash Buffer-A. After washing, the biotin-bound streptavidin was detected by adding the substrates for the alkaline phosphatase. Dissolve 50 μl BCIP stock solution (25 mg 5-bromo-4-chloro-indol-3-yl-phosphate in 500 μl dimethylformamide and store protected from light at 4 ° C) and 100 μl NBT stock solution (50 mg nitroblue tetrazolium chloride in 700 μl dimethylformamide and 300 μl of water and store protected from light at 4 ° C) are diluted in 15 ml of staining buffer (100 mM Tris-HCl, 100 mM NaCl, 5 mM MgCl ₂ , pH 9.5). After development of the membrane in this solution, recombinant, biotinylated protein could be detected in the total protein extract at a molecular weight of about 18.5 kDa in the form of a blue band. Total protein from the Escherichia coli strain XL1 without the plasmid pNCO-C Biotag-BS-LuSy had no staining in this regard. After staining, the membrane was washed in water and incubated for 5 min in a stop buffer (20 mM Tris-HCl, 25 mM EDTA-Na ₂ , pH 8.0).
• K) For renaturation of the insoluble biotinylated lumazine synthase fusion protein were initially soluble Protein fractions according to Example 1 L) separated.
• L) The insoluble residue from K) was dissolved in 100 mM K-phosphate buffer, pH 7.0, 6 M urea, 6 mM 5-nitro-6- (D-ribitylamino) 2,4- (1H, 3H) -pyrimidinedione and 100 mM dithiothreitol (DTE), solubilized for 24 hours with stirring at room temperature. The resulting protein solution was then diluted twice with 10 times the volume of 100 mM K phosphate buffer, pH 7.0, 1 mM 5-nitro-6- (D-ribitylamino) 2,4- (1H, 3H) -pyrimidinedione, and 1 mM DTE, dialysed at 4 ° C for 12 hours each. After centrifugation, the protein was contained in the supernatant using an ultracentrifuge concentrated (Beckman TFT 70 rotor (Sorvall SS34 rotor, 15000 rpm ^-1; 4 ° C; 20 min); 32000 Umin ^-1; 16 h; 4 ° C). The analysis of the renatured protein was carried out according to the statements J), Example 1 S) and Example 1 P). Renatured icosahedral fusion protein consisting of 60 subunits could be obtained by the above-described procedure.
• M) Proof of accessibility the biotin molecules in the native protein was done using microtiter plates and a multichannel photometer (ELISA reader). 100 μl of avidin stock solution (Sigma, Munich) with the concentration 1 mg / ml were first in 20 ml of coating buffer (20 mM Na carbonate, pH 9.6) (Standard solution). 100 μl each the standard solution were pipetted into the sample wells of the microtiter plate, which Sample bags afterwards sealed with an adhesive film and incubated overnight at 37 ° C. The solution was subsequently poured off and each well 3 times with 200 ul PBS (20 mM Na-phosphate, 130 mM NaCl, pH 7.2). The non-avidin covered plastic surface of the Sample bag was filled with 350 μl each Abdecklösung (3% milk powder in PBS) for 60 min at 37 ° C incubated. The cover solution was subsequently poured off and the microtiter plate tapped briefly. In the 1. of 8 sample bags, 100 μl of the renatured sample solution from L) were subsequently (about 1 mg / ml). Into the sample pockets 2-8 were each 50 .mu.l dilution solution (1 % Milk powder in PBS). The 8th sample bag was released. 50 μl out Sample bag 1 was filled with the 50 μl Dilution solution in Sample bag 2 diluted (10 × on and pipette off). 50 μl from sample bag 2 were then with 50 ul dilution solution Sample bag 3 diluted, etc. In the end were 50 μl out Sample bag 7 discarded, so now all sample bags 50 μl sample solution in various concentrations (log 2 dilution). Samples were covered with adhesive film and incubated at 37 ° C for 2 hours. The sample solutions were subsequently poured off, the microtiter plate knocked out briefly and the sample bags 3 × with each 350 ul wash solution (PBS). Subsequently were 15 μl Streptavidin-alkaline phosphatase (Promega, Madison, Wisconsin, USA) diluted in 20 ml dilution solution (detection solution). Each 100 μl of detection solution was added in the sample bags 1-8 pipetted and left for 1 hour at 37 ° C incubated. The detection solutions were subsequently poured off, the microtiter plate knocked out briefly and the sample bags 3 × with each 350 μl wash solution (PBS) washed. The development of the microtiter plate was carried out with addition of 150 μl substrate solution (10 mg p-nitrophenyl phosphate in 10 ml staining buffer (see J)) for the alkaline Phosphatase and measurement of absorbance at 405 nm. Since it is at the lumazine synthase around a spherical Molecule acts, lie the biotin molecules statistically on the surface distributed before. Are there too many biotinylated lumazine synthase molecules on the surface the microtiter plate (about Avidin bound to the microtiter plate), so are the lateral biotin molecules for one Binding to streptavidin no longer accessible. Only after reduction the concentration of biotinylated lumazine synthase molecules the lateral biotin molecules for one Binding to streptavidin accessible, i.e. the signal strength rises.

Labeling of the C-terminal End of the lumazine synthase from Bacillus subtilis with a reactive amino acid

The The following examples describe the introduction of reactive amino acids (lysine and cysteine) at the C-terminus of lumazine synthase. On the surface of the Although lumazine synthase are basic amino acids that may be covalent Coupling of chemical molecules are available, their accessibility however, is limited by adjacent amino acid residues. On the surface the lumazine synthase from Bacillus subtilis are not free Cysteine residues are due to their thiol group for a covalent Coupling of chemical molecules could be suitable.

to Improving accessibility, or to minimize steric hindrance, a linker (tentacle linker) between the C-terminus of lumazine synthase and the subject amino acid introduced, So that the reactive amino acids for almost any chemical coupling with foreign molecules become accessible.

Example 15

Extension of the C-terminus and introduction a terminal basic amino acid (lysine) as a basis for chemical coupling of target molecules

• A) The gene for the lumazine synthase from Bacillus subtilis was labeled with the oligonucleotides EcoRI-RBS-2 (see Example 2 A)) and C-Lys165 (5 'tat tat gga tcc tta ttt acc aga gcc acc acc aga acc acc gcc acc ttc gaa aga acg gtt taa gtt tgc cat ttc 3 '), which at its 3' end is complementary to the gene for lumazine synthase and immediately after the last coding base triplet of lumazine synthase a gene coding for the peptide (Gly) ₄ Ser (Gly ) ₃ Ser-Gly-Lys, amplified from the plasmid pNCO-BS-LuSy. Immediately behind the codon for lysine (aaa) at the amino acid position 165 is the oligonucleotide C-Lys165 a stop codon and a recognition sequence for the restriction endonuclease BamHI in the DNA sequence introduced. The PCR was carried out analogously to Example 1 A).
• B) The purification of the PCR approach was carried out according to Example 1 B) to give a DNA fragment 543 bp in length.
• C) The further procedure was carried out analogously to Example 1 B), E) to L). The plasmid pNCO-Lys165-BS-LuSy is obtained.
• D) The review of Expression rate or the size of the monomer Protein strand was analogous to Example 1 M). In the raw extract of Strain XL1-pNCO-Lys165-BS-LuSy could produce a protein band with a Molecular weight of about 17 kDa can be observed in a Comparison strain without pNCO-Lys165-BS-LuSy expression plasmid not was seen. The observed band corresponded to a share of approx. 10% based on all soluble Proteins (estimated).
• E) The further analysis was carried out according to Example 1 N) to Q), wherein no significant differences to the wild-type enzyme (BS-LuSy) too were watching.

Example 16

Extension and introduction of a Terminal cysteine residue to the C-terminus of lumazine synthase as a basis for the chemical coupling of target molecules

• A) The construction was carried out analogously to Example 15 A) to C), but instead of the oligonucleotide C-Lys165, the oligonucleotide C-Cys167 (5 'tat tat gga tcc tta gca gcc acc acc aga gcc acc acc ag acc acccc acc ttc gaa aga acg gtt taa gtt tgc cat ttc 3 '), which at its 3' end is complementary to the gene for lumazine synthase and immediately after the last coding base triplet of lumazine synthase a gene coding for the peptide (Gly) ₄ Ser- (Gly ) ₃ Ser (Gly) ₃ Cys, introduced. Immediately after the codon for cysteine 167 (tgc), the oligonucleotide C-Cys167 is used to introduce a stop codon and a recognition sequence for the restriction endonuclease BamHI into the DNA sequence. A DNA fragment 549 bp in length was obtained. Escherichia coli strain XL1-pNCO-Cys167-BS-LuSy was obtained.
• B) When checking the molecular mass of the monomeric protein strand (Cys167-BS-LuSy) analogous to Example 15 D) could a Band at about 17.1 kDa are observed. The watched gang corresponded to a share of about 5% based on all soluble Proteins (estimated).
• C) The further analysis was carried out according to Example 1 N) to Q), wherein no significant differences to the wild-type enzyme (BS-LuSy) too were watching.

Example 17

Coupling of a 12 amino acids long Peptide having the recognition sequence for a monoclonal antibody (anti-FLAG-M2 / IgG1 / mouse) to the N-terminus of lumazine synthase from Bacillus subtilis

• A) The gene for lumazine synthase from Bacillus subtilis was transfected with the oligonucleotides FLAG-BS-LuSy-1 (5 'ata ata ata aag ctt atg atat ata caa gga aat tta g 3 '), which at its 3' end is complementary to the 5 'end of the lumazine synthase gene from Bacillus subtilis and at the 5 'end a recognition sequence for introduces the restriction endonuclease HindIII and flag-BS-LuSy-2 (5 'tat did gaa ttc tta ttc gaa aga acg gtt taa g 3 '), which at its 3'-end komplemetär to the 3'-end of the Lumazine synthase gene from Bacillus subtilis and at the 5 'end a recognition sequence for the Restriction endonuclease EcoRI, from the plasmid pRF2 (see. Example 1 A)) amplified.
• B) The PCR approach was as in Example 1 B) to give a 492 bp length of DNA fragment, cleaned.
• C) The isolated DNA fragment and the vector pFLAG-MAC (Eastman Kodak Company, New Haven) were first digested with the restriction endonuclease HindIII. 30.0 μl pFLAG-MAC [5 μg] or 30 μl isolated DNA fragment 3.0 μl HindIII [60 U] 10.0 μl OPAU (10 ×) 57.0 μl H ₂ O _bidist. The _mixture also became 180 min incubated at 37 ° C and the resulting DNA fragment as described in Example 1 B) purified and used for digestion with the restriction endonuclease EcoRI.
• D) The isolated DNA fragment and the vector pFLAG-MAC from C) were then digested with the restriction endonuclease EcoRI. 30.0 μl DNA fragments from C) 3.0 μl EcoRI [60 U] 24.0 μl OPAU (10 ×) 63.0 μl H ₂ O _bidist. The _mixture was incubated for 180 min at 37 ° C. and purified as described under B) and used for ligation.
• E) The further procedure was carried out according to Example 1 G) to L).
• F) After completion a denaturing polyacrylamide gel electrophoresis according to Example 1 M) could in the crude extract a protein band with a molecular weight of about 17.7 kDa are observed in a control strain without pFLAG-MAC-BS-LuSy expression plasmid not to be seen. The observed gang corresponded to a share of about 10% based on all soluble Proteins (estimated).
• G) The review of the functionality was carried out analogously to Example 1 N) to P), with no significant difference to the wild-type enzyme could be observed.
• H) For immunological testing of the functionality of the FLAG peptide, a Western blot analysis according to Example 1 Q), but using the monoclonal antibody 'Anti-FLAG ^® M2' (Eastman Kodak Company, New Haven) as the 1st antibody ( 10 μl of anti- ^FLAG® M2 in 5 ml TBS (50 mM Tris, 150 mM NaCl, pH 7.4) and anti-mouse IgG-HRP conjugate as second antibody (10 μl anti-mouse IgG HRP). Conjugate in 5 ml TBS, see Example 18 H)). After development of the membrane, the fusion protein could be detected at about 17.7 kDa.
• I) The purification was carried out analogously to Example 2 S), but it became no lysozyme to the digestion buffer added.
• J) Negative contrasting on the electron microscope according to the example 1 P) showed hollow, spherical Particles with an outer diameter of about 15 nm and an inner diameter of about 5 nm.
• K) The review of quaternary structure was carried out analogously to Example 1 S). The purified lumazine synthase fusion protein was seen as a discrete band on the native gel. It could be one reduced mobility compared to a wild-type lumazine synthase, on the enlarged diameter is due to be watched.

Example 18

Coupling of a 6 amino acids long Peptides (6 × histidine) for binding to Ni-chelate affinity matrix, for binding a monoclonal antibody (penta-His) or to Binding of Ni-NTA-HRP conjugate to the C-terminus of lumazine synthase from Bacillus subtilis

• A) The gene for lumazine synthase from Bacillus subtilis was first with the oligonucleotides RibH-1 (see Example 1 A)) and RibH-His6-C-1 (5 'gtg gtg atg gtg atg ttc gaa aga acg gtt taa g 3 '), which at its 3' end completes the 3 'end of the lumazine synthase gene from Bacillus subtilis and at the 5 'end for one Part of the affinity peptide coded, from the plasmid pRF2 (see Example 1 A)) amplified.
• B) The PCR approach was as in Example 1 B) to give a 492 bp length of DNA fragment, cleaned and used as a template for a 2nd PCR according to example 1 C), but with the oligonucleotide combination EcoRI-RBS-2 (cf. Example 2 A)) and RibH-His6-C-2 (5 'did did gga tcc tta atg gtg gtg atg gtg atg 3 '), which completes the His6 domain in the 3' direction, immediately behind the last coding base triplet of this domain, a stop codon in the sequence introduces and immediately after this stop codon, a restriction endonuclease recognition sequence BamHI involves, was used.
• C) The PCR batch from B) was purified analogously to Example 1 B) and a DNA fragment of length Received 528 bp.
• D) Further processing was carried out analogously to Example 1 E) to L), wherein the Escherichia coli expression strain XL1-pNCO-C-His6-BS-LuSy was obtained.
• E) After completion a denaturing polyacrylamide gel electrophoresis according to Example 1 M) could in the crude extract a protein band with a molecular weight of about 17.1 kDa are observed in a control strain was not seen without pNCO-C-His6-BS-LuSy expression plasmid. The observed band corresponded to a share of about 30% on all soluble Proteins (estimated).
• F) The review of the functionality was carried out analogously to Example 1 N) to P), with no significant difference to the wild-type enzyme could be observed.
• G) For immunological testing of the functionality of the His6 peptide, a Western Blot analysis according to Example 1 Q), but using the monoclonal antibody 'Penta-His ^™ Antibody' (Quiagen, Hilden) as the 1st antibody (10 .mu.l penta -His ^™ Antibody in 5 ml TBS (see Example 17 H)) and anti-mouse IgG-HRP conjugate as the second antibody (10 μl anti-mouse IgG-HRP conjugate in 5 ml TBS). After development of the membrane, the fusion protein could be detected at approximately 17.1 kDa.
• H) The detection of the accessibility of the His6 peptides in the native protein was carried out using microtiter plates and a multichannel photometer (ELISA-reader). 100 μl of the crude extract solution from D) (about 5-8 mg / ml total protein) were added to the 1st of 8 sample bags. Into each of the sample bags 2-8, 50 μl dilution solution (1% milk powder in PBS) was added. The 8th sample bag was released. 50 μl from sample bag 1 were diluted with the 50 μl dilution solution in sample bag 2 (pipette up and down 10 ×). 50 .mu.l from sample bag 2 were then diluted with 50 .mu.l dilution solution from sample bag 3, etc .. Finally, 50 .mu.l were discarded from sample bag 7, so that now all sample bags 50 ul sample solution in different concentrations (log 2 dilution) contained. The samples were covered with adhesive film and incubated overnight at 37 ° C. The plastic surface of the sample bag, which was not covered with fusion protein, was incubated with 350 μl cover solution (3% milk powder in PBS) for 60 min at 37 ° C. The covering solution was then poured off, the microtiter plate was tapped out briefly and the sample pockets were washed 3 × with 350 μl of washing solution (PBS) each time. Subsequently, 10 μl of Penta-His ^™ Antibody (Quiagen, Hilden) were diluted in 5 ml of dilution solution (1st antibody). In each case 50 μl of the 1st antibody were pipetted into the sample pockets 1-8 and incubated at 37 ° C. for 2 hours. The 1st antibody was then poured off, the microtiter plate was tapped out briefly and the sample pockets were washed 3 × with 350 μl of washing solution (PBS) each time. This was followed by the addition of 150 μl of the second antibody (10 μl of anti-mouse IgG-HRP conjugate in 5 ml dilution solution, Sigma, Munich) with an incubation time of 2 h at 37 ° C. The second antibody was then poured off, the microtiter plate was tapped out briefly and the sample pockets were washed 3 × with 350 μl of washing solution (PBS) each time. The development of the microtiter plate was carried out with addition of 150 μl of substrate solution (100 mg of o-phenylenediamine in 25 ml of substrate buffer, 50 mM citric acid, pH 5) for the peroxidase and measurement of the absorbance at 492 nm. The fusion protein could be separated from the highly specific penta-His. Antibodies are detected. A concentration-dependent (log2 dilution of bound fusion protein) signal reduction could be observed.
• I) Negative contrasting on the electron microscope according to the example 1 P) showed hollow, spherical Particles with an outer diameter of about 15 nm and an inner diameter of about 5 nm.
• J) The review of quaternary structure was carried out analogously to Example 1 S). The purified lumazine synthase fusion protein was seen as a discrete band on the native gel. It could be one reduced mobility compared to a wild-type lumazine synthase, on the enlarged diameter is due to be watched.

Example 19

Production of mixtures from C-Biotag-BS-LuSy and C-His6-BS-LuSy by way of controlled in vitro refolding the insoluble proteins

• A) The cultivation of the Escherichia coli strain XL1-pNCO-C biotag BS-LuSy (Example 14) was carried out according to the example 1 K), but in a volume of 500 ml.
• B) The cultivation of the Escherichia coli strain XL1-pNCO-C-His6-BS-LuSy (Example 18) was also carried out according to Example 1 K), but in a volume of 500 ml.
• C) The digestion of the cells from A) was carried out using an ultrasound generating apparatus (Branson Sonifier B-12A, Branson Sonic Power Company, Dunbury, Connecticut). For digestion, the washed cell pellet was suspended in 40 ml separation buffer (50 mM Tris pH 9.5) and cooled on ice for 10 min. The cell suspension was then treated 15 times with the needle probe of the ultrasound machine at level 5. Subsequently, the suspension was cooled on ice for 5 min. The process was repeated a total of 4 times. The cell suspension was then centrifuged (Sorvall centrifuge with SS34 rotor; 5,000 rpm ^-1, 4 ° C, 10 min), the supernatant is separated (crude extract A-1) and further processed, the cell pellet (residue A-1).
• D) The digestion process was done a second time, the residue A-1 from C) was again suspended in 40 ml of separation buffer. The further implementation was carried out analogously to C). It became crude extract A-2 and insoluble Residue A-2 received.
• E) The digestion of cells from B) was also carried out using an ultrasound generating apparatus (Branson Sonifier B-12A, Branson Sonic Power Company, Dunbury, Connecticut). For digestion, the washed cell pellet was suspended in 40 ml separation buffer (50 mM Tris pH 9.5) and cooled on ice for 10 min. The cell suspension was then treated 15 times with the needle probe of the ultrasound machine at level 5. Subsequently, the suspension was cooled on ice for 5 min. The process was repeated a total of 4 times. The cell suspension was then centrifuged (Sorvall centrifuge with SS34 rotor, 15,000 rpm ^-1 , 4 ° C, 30 min), the supernatant B separated and processed.
• F) The insoluble Residue A-2 (C-Biotag-BS-LuSy) from D) was dissolved in 40 ml of solubilization buffer (50 mM Tris, pH 9.5, 6 M guanidinium thiocyanate (G-SCN), 100 mM dithiothreitol (DTE)) while With stirring for 24 h solubilized at room temperature.
• G) To soluble supernatant-B (40 ml) were added 6 M G-SCN and 100 mM DTE and added with stirring for 24 h Room temperature incubated.
• H) The solubilization of F) was subjected to centrifugation (Sorvall SS34 rotor; min 20; 15000 Umin ^-1; 25 ° C) to obtain residue A-3 and supernatant A-3.
• I) Subsequently were portions of supernatant A-3 and residue A-3 analyzed using SDS-polyacrylamide gel electrophoresis.
• J) The review of the supernatants was carried out analogously to Example 1 M).
• K) To check residue A-3, a spatula tip of the residue was suspended with 200 μl of protein application buffer (see Example 1 M)) and boiled for 15 min on a boiling water bath. After cooling the remaining insolubles of the sample were pelleted in an Eppendorf centrifuge (15000 rpm ^-1, 5 min, 4 ° C) and 6 ul of the clear supernatant to the stacking gel applied. The further procedure was carried out analogously to Example 1 M).
• L) From J) and K) it was found that the insoluble protein Solubilize C-Biotag-BS-LuSy to approximately 80% under the conditions indicated leaves.
• M) The residue A-3 obtained from H) was again subjected to steps F) and H) to K). The solubilization rate could not be improved.
• N) The amounts of fusion protein of the two supernatants A-3 and B were comparable.
• O) 2 ml of supernatant C-Bio-BS-LuSy (Supernatant-A-3) and 2 ml of supernatant C-His6-BS-LuSy (Supernatant B) were mixed (mixture A).
• P) 3.5 ml of supernatant C-Bio-BS-LuSy and 0.5 ml of supernatant C-His6-BS-LuSy were mixed (mixture B).
• Q) Mixture A and Mixture B were at room temperature for 48 h touched.
• R) Mixture A and Mixture B from Q) were then used against 400 ml separation buffer containing 8 M urea and 1 mM DTE during 18 h dialyzed at room temperature.
• S) To both mixtures was then added 6.6 mM 5-nitro-6- (D-ribitylamino) 2,4- (1H, 3H) -pyrimidinedione and stirred for 8 h at room temperature and mixture A-nitro or mixture B-nitro.
• T) mixture A-nitro and mixture B-nitro were transferred to a dialysis tube and against 32 ml (5-fold volume) refolding buffer A (100 mM K-phosphate buffer, pH 7.0, 1 mM 5-nitro-6- (D-ribitylamino) 2,4- (1H, 3H) -pyrimidinedione, 1 mM DTE, 0.02% Na azide) 12 hours at 4 ° C dialyzed and obtained mixture A-1/5 and mixture B-1/5.
• U) Subsequently the dialysis buffer from T) was mixed with 40 ml refolding buffer B (100 mM K-phosphate buffer, pH 7.0, 1 mM DTE, 0.02% Na azide) and dialysed for a further 24 h at 4 ° C (Mixture A-1/10 and mixture B-1/10).
• V) Subsequently the dialysis buffer from U) was diluted with 80 ml refolding buffer B and another 24 h at 4 ° C dialysed and the dialysate mixture A-1/20 and mixture B-1/20 receive.
• W) mixture A-1/20 and mixture B-1/20 were then against 72 ml (10-fold volume) refolding buffer C (100 mM K-phosphate buffer, pH 7.0, 1 mM DTE, 0.25 mM 5-nitro-6- (D-ribitylamino) 2,4- (1H, 3H) -pyrimidinedione, 0.02% Na azide) at 4 ° C for 24 h dialyzed and obtained mixture A-1/200 and mixture B-1/200.
• X) After centrifugation (Sorvall SS34 rotor; min 20;; 15000 Umin ^-1 4 ° C) were portions of supernatant mixture A-1/200 or mix B-1/200 and residue mixture A-1/200 or mixture B-1/200 using SDS-polyacrylamide gel electrophoresis.
• Y) The check of the supernatants was carried out analogously to Example 1 M). Z) The review of Residues took place analogous to K).
• AA) From Y) and Z) it was found that about 50-80% mixed Produce lumazine synthase conjugate under the specified conditions leaves. The Fusion protein amounts of the protein amounts used corresponded in the refolded Condition (estimated from SDS-PAGE), so that no Difference in the refolding behavior the two fusion proteins could be detected.
• BB) The review of the quaternary structure was carried out analogously to Example 1 S), but with 40 ul protein solution (supernatant mixture A-1/200, supernatant mixture B-1/200) was used. The renatured fusion protein mixtures were seen as discrete bands on the native polyacrylamide g. The mobility was comparable to the mobilities the renatured proteins C-Bio-BS-LuSy and C-His6-BS-LuSy, most likely on the similar hydrodynamic diameter of the proteins is due. With the above measure could therefore renatured, icosahedral, from 60 subunits existing fusion protein can be obtained.
• CC) The detection of the two subunits in the renatured icosahedral protein was carried out using microtiter plates and a multichannel photometer (ELISA reader). The coating of the wells with avidin was carried out analogously to Example 14 M). 100 μl of the renatured sample solution from W) (about 0.5-1 mg / ml) were added to the 1st of 8 sample bags. Into each of the sample bags 2-8, 50 μl dilution solution (1% milk powder in PBS) was added. The 8th sample bag was released. 50 μl from sample bag 1 were diluted with the 50 μl dilution solution in sample bag 2 (pipette up and down 10 ×). 50 .mu.l from sample bag 2 were then diluted with 50 .mu.l dilution solution from sample bag 3, etc .. Finally, 50 .mu.l were discarded from sample bag 7, so that now all Probenta 50 μl of sample solution in various concentrations (log 2 dilution). The samples were covered with adhesive film and incubated overnight at 37 ° C, after which they were washed 3x with each 350 μl of washing solution (PBS). Subsequently, 10 μl of Penta-His ^™ Antibody (Quiagen, Hilden) were diluted in 5 ml of dilution solution (1st antibody). In each case 50 μl of the 1st antibody were pipetted into the sample pockets 1-8 and incubated at 37 ° C. for 2 hours. The 1st antibody was then poured off, the microtiter plate was tapped out briefly and the sample pockets were washed 3 × with 350 μl of washing solution (PBS) each time. Subsequently, 150 μl of the second antibody (10 μl of anti-mouse IgG-HRP-HRP conjugate in 5 ml dilution solution, Sigma, Munich) were added with an incubation time of 2 h at 37 ° C. The second antibody was then poured off, the microtiter plate was tapped out briefly and the sample pockets were washed 3 × with 350 μl of washing solution (PBS) each time. The development of the microtiter plate was carried out with the addition of 150 μl substrate solution (100 mg o-phenylenediamine in 25 ml substrate buffer, 50 mM citric acid, pH 5) for the peroxidase and measurement of the absorbance at 492 nm. Supernatant mixture A-1/200 and supernatant Mixture B-1/200 could be recognized by the highly specific penta-His antibodies. A concentration-dependent (log2 dilution of bound fusion protein) signal reduction could be observed. In supernatant mixture A-1/200 a stronger signal than in supernatant mixture B-1/200 could be observed. For binding to the avidin-coated microtiter plate theoretically a biotin molecule is sufficient, while the signal strength correlates with the number of His6 epitopes per molecule. In supernatant mixture A-1/200, more His6 epitopes are contained per icosahedron than in the comparative construct supernatant mixture B-1/200, so that in supernatant mixture A-1/200, as the practical experiment confirms, a stronger signal is to be expected. From this result it follows that with the refolding measures described above, icosahedra consisting of different subunits have been obtained.

Example 20

Preparation of the thermostable Lumazine synthase from Aquifex aeolicus using 11, to the Escherichia coli codon usage adapted synthetic Oligonucleotides and a 6-stage polymerase chain reaction (Deckert et al., 1998)

• A) The first part of the gene for the lumazine synthase from Aquifex aeolicus was with the oligonucleotides AQUI-1 (5'gct gcg gta gaa gt ctg gcg cgt aaa gag gac gat gct Gtt gc gc gc gtt cc gc ctc ctc atc 3 ', strand primer) and AQUI-2 (5'cta atg aaaa ggt tcg cga ggc ctt ttg aaa ctt cag agg cga tat aat cga aat gtg gcg ttg 3 '; opposite strand primer) using the gene for lumazine synthase from Bacillus subtilis as template (vector pNCO-BS) LuSy, see Example 1 G)). Both oligonucleotides were adapted for optimal codon usage (for strongly expressed genes, Grosjean and Fiers, 1982, Ikemura, 1981, Wada et al., 1992) by Escherichia coli. AQUI-1 contains the recognition sequence for the restriction endonuclease MfeI (C * AATTG) and AQUI-2 for StuI (AGG * CCT). 10 μl of PCR buffer (75 mM Tris / HCl, pH 9.0, 20 mM (NH ₄ ) ₂ SO ₄ 0.01% (w / v) Tween 20) 6 μl Mg ²⁺ [1.5 mM] 8 μl dNTP's [je 200 μM] 1 μl AQUI-1 [0.5 μM] 1 μl AQUI-2 [0.5 μM] 1 μl pNCO-BS-LuSy (see Example 1) [10 ng] 1 μl Goldstar Taq polymerase [ 0.5 U] (Eurogentec, Seraing, Belgium) 72 ul H ₂ O _bidest The batch was in the thermocycler (GeneAmp ^® PCR system 2400; Perkin Elmer) under the following conditions: 1. 95 ° C 5.0 min 2. 0 , 5 min 94 ° C 3. 0.5 min 50 ° C 4. 0.5 min 72 ° C 5. 5.0 min 72 ° C 6. Cool to 4 ° C Steps 2.-4. were repeated 20 times.
• B) The PCR batch was analyzed by agarose gel electrophoresis (3 % Agarose), the DNA by staining with ethidium bromide under UV light visualized and the fragment with a length of Cut 132 bp out of the gel with a scalpel. The others Purification was carried out according to Example 1 B).
• C) 10 ng of the isolated DNA from B) then served as template for a 2.PCR with the oligonucleotides AQUI-3 (5 'act ctg gtt cgt gtt cca ggc tca tgg gaa ata ccg gtt gct gcg ggt gaa ctg gcg cgt aaa g 3 ', strand primer) and AQUI-4 (5' cca agg tgt cag ctg taa taa cac cga agg tga tag gtt tac gta gtt cta atg aaa ggt tcg cga ggc c 3 '; Strand primer). Both oligonucleotides were adapted for optimal codon usage for highly expressed genes (Grosjean and Fiers, 1982, Ikemura, 1981, Wada et al., 1992) by Escherichia coli. AQUI-3 contains the recognition sequence for the restriction endonuclease AgeI (A * CCGGT) and AQUI-4 for SnaBI (TAC * GTA) and PvuII (CAG * CTG). The polymerase chain reaction was carried out as described under A).
• D) The product from C) was purified as described under B) and a DNA fragment of length Received 219 bp.
• E) 10 ng of the isolated DNA from D) then served as Matrix for a 3.PCR with the oligonucleotides AQUI-5 (5 'gga ggg tgc aat tga ttg cat agt ccg tca tgg cgg ccg tga aga aga cat tac tct ggt tcg tgt tcc agg c 3 '; Strand primer) and AQUI-6 (5 'gtt gcc gtg ttt tgt gcc ggc gcg ctc gat agc ctg ttc caa ggt gtc agc tgt aat aac 3 '; Strand primer). Both oligonucleotides were assigned to the optimal codon usage (for strongly expressed Genes) of Escherichia coli. AQUI-5 contains the recognition sequence for the Restriction endonuclease EagI (C * GGCCG) and AQUI-6 for BssHII (G * CGCGC) and PvuII (CAG * CTG). The polymerase chain reaction took place as described under A).
• F) The product of E) was purified as described under B) and a DNA fragment of length Received 309 bp.
• G) 10 ng of the isolated DNA from F) subsequently served as Matrix for a 4.PCR with the oligonucleotides AQUI-7 (5 'cgg act cgt agc atc acg tttta ta tca tgc tct tgt cga ccg tct ggt gga ggg tgc aat tga ttg cat ag 3 '; Strand primer) and AQUI-8 (5 'gaa taa gtt tgc cat ttc aat ggc aga aag cgc tgc ttc cca acc ttt gtt gcc gtg ttt tgt gcc ggc 3 '; Strand primer). Both oligonucleotides were given the optimal Codon usage (for strong expressed genes) of Escherichia coli. AQUI-7 contains the recognition sequence for the Restriction endonuclease SalI (G * TCGAC) and AQUI-8 for Eco47III (AGC GCT *). The polymerase chain reaction was carried out as described under A).
• H) The product from G) was purified as described under B) and a DNA fragment of length 405 bp received.
• I) 10 ng of the isolated DNA from H) then served as Matrix for a 5. PCR with the oligonucleotides AQUI-9 (5 'atg caa atc tac gaa ggt aaa cta act gct gaa ggc ctt cgt ttc ggt atc gta gca tca cgt ttt aat c 3 '; Strand primer) and AQUI-10 (5 'did did gga tcc tta tcg gag aga ctt gaa taa gtt tgc cat ttc aat gg 3 '; Strand primer). Both oligonucleotides were assigned to the optimal codon usage (for strong expressed genes) of Escherichia coli. AQUI-9 completes that Gene for the lumazine synthase from Aquifex aeolicus and contains the recognition sequence for the Restriction endonuclease StuI (AGG * CCT). The oligonucleotide AQUI-10, which at the 3'-end to the gene of lumazine synthase from Aquifex aeolicus is complementary, leads directly behind the stop codon a recognition sequence for the endonuclease BamHI (G * GATCC) one. The polymerase chain reaction was carried out as described under A).
• J) The product from I) was purified as described under B) and a DNA fragment of a length received from 476 bp.
• K) 10 ng of the isolated DNA from 1) subsequently served as Matrix for a 6.PCR with the oligonucleotides AQU1-11 (5 'ata ata gaa ttc att aaa gag gag aaa tta act atg caa atc tac gaa ggt aaa cta ac 3 '; Strand primer) and AQUI-10 (antisense primer). The oligonucleotide AQUI-11 is at the 3 'end to the 5' end of the synthetic gene for lumazine synthase from Aquifex aeolicus complementary. At the 5'-end it performs a ribosomal Binding site and a recognition sequence for the restriction endonuclease EcoRI before the start codon. The polymerase chain reaction took place as described under A).
• L) The product from K) was purified as described under B) and a DNA fragment of length Received 510 bp.
• M) The further processing of the DNA fragment from L) was carried out as in Example 1 E) -G) wherein the plasmid pNCO-AA-LuSy was obtained.
• N) Subsequently were the steps H) -M) according to example 1 performed. In the crude extract of the strain XL1-pNCO-AA-LuSy a protein band could be detected with a molecular weight of 16.7 kDa, which in a reference strain without the relevant plasmid to see was. The observed band corresponded to a share of about 20% on all soluble Proteins.
• O) The review of functionality took place according to example 1N). The protein showed optimal activity in the range of 80-90 ° C.
• P) The negative contrast was carried out according to Example 1 P) and showed hollow, spherical Particles with an outer diameter of about 15 nm and an inner diameter of about 5 nm.
• Q) Purification of the protein took place in two steps: Cultivation of the Escherichia coli strain XL1-pNCO-AA-LuSy was carried out according to Example 1 K), but in a volume of 1 l. The digestion was carried out according to Example 1 R). The clear supernatant was then incubated in a water bath at 90 ° C for 20 min. The suspension was then centrifuged (Sorvall SS34 rotor, 15000 rpm ^-1; 4 ° C; 30 min). With this step, an enrichment of the target protein by factor 4 could be achieved (estimated by SDS-PAGE). Further purification was carried out according to Example 2 S) using the supernatant centrifugation and a gel filtration column.
• R) The review of the quaternary structure took place according to example 1 S) and gave one with the lumazine synthase from Bacillus subtilis comparable result.

Example 21

Coupling of an artificial 13 amino acids long, in vivo biotinylatable Peptides, to the C-terminus of the thermostable lumazine synthase Aquifex aeolicus

• A) The gene for synthetic lumazine synthase from Aquifex aeolicus was prepared using the polymerase chain reaction and the synthetic oligonucleotides EcoRI-RBS-2 (see example 2 A)) and AQUI-C-NotI (5 'did did did agc ggc cgc tcg gag aga ctt gaa taa g 3 ') from the plasmid pNCO-AA-LuSy (see example 20) amplified. The oligonucleotide AQUI-C-NotI is complementary to the gene at its 3 'end for the Lumazine synthase and leads immediately after the last coding base triplet of lumazine synthase a recognition sequence for the restriction endonuclease NotI (GC * GGCCGC). This recognition sequence becomes in three Alaninmoleküle translated. The implementation the PCR was carried out analogously to Example 1 A).
• B) The PCR approach was as in Example 1 B) to give a DNA fragment of 513 bp in length, cleaned.
• C) The purified DNA fragment from B) was digested with the restriction endonuclease NotI analogous to Example 8 F) digested and then purified according to Example 1 B).
• D) The purified DNA fragment from C) was analogous to Example 8 H) treated with the restriction endonuclease EcoRI and a fragment with the length received from 498 bp.
• E) 5 μg of the expression plasmid pNCO-C-Biotag-BS-LuSy (Example 14) in one Volume of 30 μl were treated analogously to Example 8 G) and H). The DNA fragment with a length of 3437 bp was isolated and purified.
• F) Further processing was carried out analogously to Example 1 E) to L), wherein the Escherichia coli expression strain XL1-pNCO-C-Biolag-AA-LuSy was obtained.
• G) When checking the functionality the lumazine synthase according to Example 1 N) could only have one enzymatic activity, that of the activity of Escherichia coli strain XL1.
• H) The review of the Expression rate or the size of the soluble Proteins were carried out analogously to Example 1 M). In the raw extract of the strain XL1-pNCO-C-Biolag-AA-LuSy failed to produce any significant protein band be observed with a molecular weight of about 18.5 kDa be, the in a control strain without pNCO-C Biolag AA-LuSy expression plasmid not to be seen.
• I) Further processing was carried out analogously to Example 14 H) to M). A comparable result could be achieved.

Example 22

Coupling of an artificial 13 amino acids long, in vivo biotinylatable Peptides, about a linker consisting of the amino acid sequence H-H-H-H-H-A-A-A to the C-terminus of the thermostable lumazine synthase from Aquifex aeolicus

• A) The gene for the synthetic lumazine synthase from Aquifex aeolicus was analogous to Example 21 using the polymerase chain reaction and the synthetic oligonucleotides EcoRI-RBS-2 (see Example 2 A)) and AQUI-C-HIS ₆ -NotI (5 'tat did tat agc ggc cgc atg gtg gtg atg gtg atg tcg gag aga ctt gaa taa gtt tgc 3 ') from the plasmid pNCO-AA-LuSy (see Example 20) amplified. The oligonucleotide AQUI-C-HIS ₆ -NotI is complementary at its 3 'end to the gene for lumazine synthase and leads immediately after the last coding base triplet of lumazine synthase a sequence coding for 6 histidine molecules and a recognition sequence for the restriction endonuclease NotI (GC * GGCCGC). This recognition sequence is translated into three alanine molecules. The PCR was carried out analogously to Example 1 A).
• B) The PCR approach was as in Example 1 B) to give a DNA fragment 531 bp in length, cleaned.
• C) The purified DNA fragment from B) was digested with the restriction endonuclease NotI analogous to Example 8 F) digested and then purified according to Example 1 B).
• D) The purified DNA fragment from C) was analogous to Example 8 H) treated with the restriction endonuclease EcoRI and a fragment with the length received from 516 bp.
• E) The expression vector was prepared according to Example 21 E).
• F) Further processing was carried out analogously to Example 1 E) to J), wherein the Escherichia coli expressions XL1-pNCO-HIS6-C Biotag-AA-LuSy was obtained.
• G) The cultivation of the cells was carried out according to Example 1 R). After centrifugation, the clear supernatant was discarded and washed with the remaining residue continue working.
• H) The insoluble residue from G) was stirred in 50 ml of NTA buffer A (50 mM Na phosphate buffer pH 8.0, 300 mM NaCl, 0.02% Na azide, 6 M guanidinium hydrochloride) for 24 hours incubated at room temperature. This was followed by centrifugation (Sorvall SS34 rotor, 15000 rpm ^-1, 20 ° C, 20 min). The supernatant was decanted and treated with 6 ml of Ni-NTA agarose (Quiagen, Hilden) and incubated overnight (20 ° C.) while swirling. Subsequently, the suspension was subjected to centrifugation (800 g, 20 ° C, 10 min). The supernatant was decanted. The residue was taken up in 10 ml of NTA buffer-A and incubated at 20 ° C. for 15 minutes while inverting. After centrifugation (see above) and removal of the supernatant, the residue was incubated in 10 ml NTA buffer B (8 M urea, 100 mM Na phosphate buffer, 10 mM Tris pH 6.3) for 15 min at 20 ° C and further processed (see above). The process was repeated a second time. The residue was then treated twice with 10 ml of NTA buffer C (8 M urea, 100 mM Na phosphate buffer, 10 mM Tris pH 5.9). Finally, the residue was washed twice with 10 ml each of NTA buffer D (8 M urea, 100 mM Na phosphate buffer, 10 mM Tris pH 4.5). The contaminants could be removed with NTA buffer B, the target protein was eluted with NTA buffer C and D, respectively. On a denaturing polyacrylamide gel (after neutralization) according to Example 1 M) only a single band at 19.3 kDa could be observed.

Example 23

Coupling of an artificial 13 amino acids long, in vivo biotinylatable Peptides, about a linker consisting of the amino acids H-H-H-H-H-G-G-S-G-A-A-A to the C-terminus of the thermostable lumazine synthase from Aquifex aeolicus

• A) The gene for the synthetic lumazine synthase from Aquifex aeolicus was prepared analogously to Example 21 using the polymerase chain reaction and the synthetic oligonucleotides EcoRI-RBS-2 (see Example 2 A)) and AQUI-C-HIS ₆ -GLY ₂ -SER-GLY- NotI (5 'did did agc ggc cgc gcc aga acc gcc atg gtg gtg atg gtg atg tcg gag aga ctt gaa taa gtt tgc 3') from the plasmid pNCO-AA-LuSy (see example 20). The oligonucleotide AQUI-C-HIS ₆ -GLY ₂ -SER-GLY-NotI is complementary at its 3 'end to the gene for the lumazine synthase, immediately after the last coding base triplet of the lumazine synthase a sequence coding for the peptide HHHHHHGGSG, and a recognition sequence for the restriction endonuclease NotI (GC * GGCCGC). This recognition sequence is translated into three alanine molecules. The PCR was carried out analogously to Example 1 A).
• B) The PCR approach was as in Example 1 B) to give a DNA fragment 543 bp in length, cleaned.
• C) The purified DNA fragment from B) was digested with the restriction endonuclease NotI analogous to Example 8 F) digested and then purified according to Example 1 B).
• D) The purified DNA fragment from C) was analogous to Example 8 H) treated with the restriction endonuclease EcoRI and a fragment with the length received from 528 bp.
• E) The expression vector was prepared according to Example 21 E).
• F) Further processing was carried out analogously to Example 1 E) to L), wherein the Escherichia coli expression strain XL1-pNCO-HIS6-GLY2-SER-GLY-C-Biotag-AA-LuSy was obtained.
• G) The further processing was carried out according to Example 22 G) to H) where a comparable result was achieved.

Example 24

Production of a chimeric protein consisting of a part of the lumazine synthase from Bacillus subtilis and part of the thermostable lumazine synthase from Aquifex aeolicus

• A) A part of the gene for lumazine synthase from Bacillus subtle was made using the polymerase chain reaction and the synthetic oligonucleotides EcoRI-RBS-2 (see Example 2 A)) and BS-LuSy-AgeI (5 'tat did did aac cgg did ttc aaa tgc gcc 3 ') from the plasmid pNCO-BS-LuSy (see Example 1) amplified. The oligonucleotide BS-LuSy-AgeI is on its 3 'end complementary to the gene for the Lumazine synthase and leads a recognition sequence for insert the restriction endonuclease AgeI (A * CCGGT) into the DNA sequence. The implementation the PCR was carried out analogously to Example 1 A).
• B) The PCR batch was purified according to Example 1 B) to obtain a 225 bp DNA fragment. The purified DNA fragment from B) was digested with the restriction endonuclease AgeI. 30.0 μl of DNA fragments from C) 4.0 μl AgeI [8 U] 10.0 μl Buffer 1 (10 ×) [10 mM bis-tris-propane-HCl, 10 mM MgCl ₂ , 1 mM DTT, pH _7.0.0 56.0 μl H ₂ O _bidist. The _mixture was incubated for 180 min at 25 ° C. and purified as described under B) and used for digestion with the restriction endonuclease EcoRI.
• C) digestion of the purified fragment from B) with the restriction endonuclease EcoRI. 30.0 .mu.l DNA fragment from B) 3.0 .mu.l EcoRI [60 U] 20.0 .mu.l OPAU (10x) 47.0 _{.mu.l bidistilled} H ₂ O ₂ The batch was incubated for 180 min at 37 ° C. and the resulting DNA Fragment as described in Example 1 B).
• D) The plasmid PNCO-AA-LuSy (30 μl, 5 μg) was treated analogously to B) and C) and subsequently according to example 1 B), with a DNA fragment of length 3676 bp was obtained.
• E) Further processing was carried out analogously to Example 1 E) to L), wherein the Escherichia coli expression strain XL1-pNCO-BS-LuSy-AgeI-AA-LuSy was obtained.
• F) When reviewing the functionality the lumazine synthase according to Example 1 N) an enzymatic activity could be determined.
• G) The review of the Expression rate or the size of the soluble Proteins were carried out analogously to Example 1 M). In the raw extract of the strain XL1-pNCO BS lusy-AgeI AA lusy could produce a significant protein band with a molecular weight of about 16.4 kDa are observed in a control strain not to be seen without pNCO-BS-LuSy-AgeI-AA-LuSy expression plasmid was.

Example 25

Making a vector for the recombinant N-terminal fusion of foreign peptides to the lumazine synthase from Aquifex aeolicus (foreign peptides can also without linker directly to the carrier protein be merged; therefor becomes the singular Restriction site BglII used in the sequence of the carrier protein is)

• A) The gene for the lumazine synthase from Aquifex aeolicus was treated with the oligonucleotides AQUI-11-BglII (5 'ata ata gaa ttc att aa gag gag aaa tta act atg cag atc tac gaa gg 3 '), which at the 3' end is partially complementary to the 5 'end of the lumazine synthase from Aquifex aeolicus is, with a silent mutation a unique recognition sequence for the Restriction endonuclease BglII (A * GATCT) is introduced and at the 5 'end a ribosomal Binding site and a recognition sequence for the restriction endonuclease EcoRI introduces, and AQUI-10 (see Example 20 I)) from the plasmid pNCO-AA-LuSy amplified. The PCR was carried out analogously to Example 1 A).
• B) The purification of the PCR approach was carried out according to Example 1 B) to give a DNA fragment of 510 bp in length.
• C) The further procedure was carried out analogously to Example 1 B), E) to L). The plasmid pNCO-AA-BglII-LuSy is obtained.
• D) The review of Expression rate or the size of the monomer Protein strand was analogous to Example 1 M). In the raw extract of Strain XL1-pNCO-AA-BglII-LuSy could produce a protein band with a molecular weight of about 16.7 kDa are observed in a control strain was not seen without pNCO-AA-BglII-LuSy expression plasmid. The observed band corresponded to a share of about 20% based on all soluble Proteins (estimated).
• E) The further analysis was carried out according to Example 20 O) to R), wherein no significant differences to the wild-type enzyme (AA-LuSy) too were watching.

Example 26

Making a vector for the C-terminal fusion of foreign peptides to lumazine synthase from Aquifex aeolicus

• A) The gene for the lumazine synthase from Aquifex aeolicus was with the oligonucleotides EcoRI-RBS-2 (see Example 2 A)) and AQUI-10- (BamHI) (5 'tat tat gga tcc tcg gag aga ctt gaa taa gtt tgc 3 '), which is complementary to the 3' end of the lumazine synthase from Aquifex aeolicus at the 3 'end and introduces a unique recognition sequence for the restriction endonuclease BamHI (G * GATCC) immediately after the last coding base triplet (the stop codon contained in the original sequence is dissolved; as a new stop codon, a stop codon present in the vector sequence) is amplified from the plasmid pNCO-AA-LuSy. The PCR was carried out analogously to Example 1 A).
• B) The purification of the PCR approach was carried out according to Example 1 B) to give a DNA fragment of 507 bp in length.
• C) The further procedure was carried out analogously to Example 1 B), E) to L). The plasmid pNCO-AA-LuSy (BamHI) is obtained.
• D) The review of Expression rate or the size of the monomer Protein strand was analogous to Example 1 M). In the raw extract of Strain XL1-pNCO-AA-LuSy- (BamHI) could be a protein band with a Molecular weight of about 17.8 kDa are observed in a Comparison strain without pNCO-AA-LuSy (BamHI) expression plasmid not was seen. The observed band corresponded to a share of approx. 20% based on all soluble Proteins (estimated).
• E) The further analysis was carried out according to Example 20 O) to R), wherein no significant differences to the wild-type enzyme (AA-LuSy) too were watching.

Example 27

Making a vector for the simultaneous N- and C-terminal fusion of foreign peptides to the lumazine synthase from Aquifex aeolicus

• A) The gene for the lumazine synthase from Aquifex aeolicus was probed with the oligonucleotides EcoRI-RBS-2 (see example 2 A)) and AQUI-10 (BamHI) (see Example 26), from the plasmid pNCO-AA-BglII-LuSy amplified. The PCR was carried out analogously to Example 1 A).
• B) The purification of the PCR approach was carried out according to Example 1 B) to give a DNA fragment of 507 bp in length.
• C) The further procedure was carried out analogously to Example 1 B), E) to L). The plasmid pNCO-AA-BglII-LuSy (BamHI) is obtained.
• D) The review of Expression rate or the size of the monomer Protein strand was analogous to Example 1 M). In the raw extract of Strain XL1-pNCO-AA-BglII-LuSy (BamHI) could a protein band with a molecular weight of about 17.8 It can be observed in a control strain without pNCO-AA-BglII-LuSy (BamHI) expression plasmid not to be seen. The observed gang corresponded to a share of about 20% based on all soluble Proteins (estimated).
• E) The further analysis was carried out according to Example 20 O) to R), wherein no significant differences to the wild-type enzyme (AA-LuSy) too were watching.

literature

• Altschul, Stephen F., Thomas L. Madden, Alejandro A. Schäffer, Jinghui Zhang, Zheg Zhang, Webb Miller, David J. Lipman (1997) gapped BLAST and Psi-BLAST: a new generation of protein database search programs Nucleic Acids Res. 25, 3389-3402
• Bacher A. and R. Ladenstein (1991) The lumazine synthase / riboflavin synthase complex of Bacillus subtilis in Chemistry and Biochemistry of Flavoproteins, Volume 1 (F. ed.) CRC Press, Boca Raton, 293-316
• Bacher A., H.C. Ludwig, H. Schnepple and Y. Ben-Shaul (1986) Heavy riboflavin synthase from Bacillus subtilis. Quaternary structure and reaggregation. J. Mol. Biol., 187, 75-86
• Bacher A., H. Schnepple, B. Milanese, M.K. Otto and Y. Ben-Shaul (1980) Structure and function of the riboflavin synthase complex of Bacillus subtilis in Flavins and Flavoproteins, (K. Yagi and T. Yamano, eds.) Japan Scientific Societies Press, 579-586
• Bacher A., S. Eberhardt, M. Fischer, S. Mörtl, K. Kis, K. Kugelbrey, J. Scheuring and K. Schott (1997) Biosynthesis of riboflavin. Lumazine synthase and riboflavin synthase Methods Enzymol., 280, 389-399
• Birnboim H.C., J. Doly (1979) A Rapid Alkaline Extraction Procedure for Screening Recombinant Plasmid DNA Nucl. Acids. Res., 7, 1513-1522
• Bradford M. (1976) A rapid and selective method for the quantitation of microgram quantities of protein using the prinziple of protein-dye binding Anal. Biochem., 72, 248-254
• Brigidi P., E. Rossi, M. Bertarini, G. Riccardi, D. Matteuzzi (1990) Bacillus subtilis by electroporation FEMS Microbiology Letters, 67, 135-138
• Bullock W.O., J.M. Fernandez, J.M. Short (1987) XL1-Blue: a high efficiency plasmid transforming recA Escherichia coli strain with β-galactosidase selection BioTechniques, 5, 376-379
• Clackson T., D. Güssow, P. T. Jones (1991) General applications of PCR to gene cloning and manipulation In: M.J. McPherson, P. Quirke, G.R. Taylor (Eds.); PCR: A practical approach (187-214). Oxford Press
• Cohn E. and Edsall J. (1943) Protein, amino acids and peptides asions and dipolar ions Reinhold, New York
• Compton S.J., C.G. Jones (1985) Mechanism of Dye Response and Interference in the Bradford Protein Assay Annal. Biochem. 151, 369
• Cronan J.E. (1990) Biotinification of proteins in vivo The Journal of Biological Chemistry, 265/18, 10327-10333
• Dalsgaard K., Uttenthal A., Jones T., Xu F., Merryweather A., Hamilton W., Langeveld J., Boshuizen R., Kamstrup S., Lomonossoff G., Porta C., Vela C., Casal J., Meloen R., Rodgers P. (1997) Plant-derived vaccine protects target animals against a viral disease Nature Biotechnology, Volume 15, 248-252
• Davis L.G., M.D. Dibner, J.F. Battey (1986) Basic Methods in molecular biology Elsevier, New York, Amsterdam, London
• Deckert G., Warren P., Gaasterland T., Young W., Lenox A., Graham D., Overbeek R., Snead M., Keller M., Aujay M., Huber R., Feldman R., Short J., Olsen G., Swanson R. (1998) The complete genome of the hyperthermophilic bacterium Aquifex aeolicus Nature, 392, 353-358
• Dower W.J., J.F. Miller, C.W. Ragsdale (1988) High efficiency Transformation of Escherichia coli by high voltage electroporation Nucl. Acids Res., 16, 6127-6145
• Glick B.R., Pasternak J.J. (1995) monekluare biotechnology spectrum Academic Publishing, Heidelberg, Berlin, Oxford
• Grosjean H. and W. Fiers (1982) Preferential codon usage in procaryotic genes-the optimal codon-anticodon interaction energy and the selective codon usage in efficiently expressed genes genes 18, 199-209
• Hennecke H., Günther I., Binder F. (1982) A novel cloning vector for the direct selection of recombinant DNA in Escherichia coli Genes, 19, 231-234
• Henner D. (1990) Expression of Heterologous Genes in Bacillus subtilis Meth. Enzymol., 185, 199
• Ikemura T. (1981) Correlation between the abundance of Escherichia coli transfer RNAs and the occurence of the respective codons in is protein genes: A proposal for a synonymous codon choice that is optimal for the E. coli translational system J. Mol. Biol., 151, 389-409
• Ladenstein R., B. Meyer, R. Huber, H. Labischinski, K. Bartels, H.-D. Bartunik, L. Bachmann, H.C. Ludwig and A. Bacher (1986) Heavy riboflavin synthase from Bacillus subtilis. Particle dimensions, crystal packing and molecular symmetry. J. Mol. Biol., 187, 87-100
• Ladenstein R., H.D. Bartunik, M. Schneider, R. Huber, K. Schott and A. Bacher (1986) Structure of the riboflavin synthase / lumazine synthase complex. Arrangement and chain folding of β subunits in the icosahedral capsid. in Chemistry and Biology of Pteridines, (B.A. Cooper, V.M. Whitehead, eds.) Walter de Gruyter, Berlin, 103-106
• Ladenstein R., K. Ritsert, R. Huber, G. Richter and A. Bacher (1994) The lumazine synthase / riboflavin synthase complex of Bacillus subtilis: X-ray structure analysis of reconstitut ed β ₆₀ capsids at 3.2 Å resolution Eur. J. Biochem., 223, 1007-1017
• Ladenstein R., M. Schneider, R. Huber, HD Bartunik, K. Wilson, K. Schott and A. Bacher (1988) Heavy riboflavin synthase from Bacillus subtilis. Crystal structure analysis of the icosahedral β ₆₀ capsid at 3.3 Å resolution J. Mol. Biol., 203, 1045-1070
• Ladenstein R., M. Schneider, R. Huber, K. Schott and A. Bacher (1988). The structure of the icosahedral β ₆₀ capsids of heavy riboflavin synthase from Bacillus subtilis Z. Crystallography, 185, 122-124
• Laemmli U.K. (1970) Cleavage of Structural Proteins During the Assembly of the Head of Bacteriophage T4 Nature, 227, 680-685
• Laue T., Shah B., Ridgeway T., Pelletier S. (1992) computer aided interpretation of analytical sedimentation data for proteins in: Harding S., Rowe A., Horton J. (eds.) Analytical ultracentrifugation in biochemistry and polymer science Royal Society of Chemistry, Cambridge, 90-125
• LeGrice S.F.J. (1990) A regulated promoter for high level expression of heterologous genes in Bacillus subtilis Meth. Enzymol., 185, 201
• Liddell E., Weeks I. (1996) Antibody techniques spectrum Academic Publishing, Heidelberg, Berlin, Oxford
• Lottspeich F., Zorbas H. (1998) bioanalysis spectrum Academic Publishing, Heidelberg, Berlin, Oxford
• Lovett P.S. (1981) BR151ATCC 33677 - Bacillus subtilis BR151 J. Bacteriol., 146, 1162-1165
• Maniatis T., Fritsch E.F., Sambrook J. (1982) Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
• Modrow S., Hawk D. (1997) Molecular Virology spectrum Academic Publishing, Heidelberg, Berlin, Oxford
• Mörtl S., M. Fischer, G. Richter, J. Tack, S. Weinkauf and A. Bacher (1996) Biosynthesis of riboflavin. Lumazine synthase of Escherichia coli. J. Biol. Chem., 271, 33201-33207
• Mullis K., F. Faloona, S. Scharf, R. Saiki, G. Horn, H. Ehrlich (1986) Specific enzymatic amplification of DNA in vitro: The polymerase chain reaction Cold Spring Harbor Symp., 51, 263-273
• Perkins JB, JG Pero, A. Sloma (1991) Riboflavin overproducing bacteria expressing the rib-operon of Bacillus Eur. Pat. Appl. EP 405370 A1 910102
• Read S.M., D.H. Northcote (1981) Minimization of variation in the response to different proteins of the Coomassie Blue G dyebinding assay for protein Anal. Biochem., 116, 53-64
• Sambrook J., E. Fritsch, T. Maniatis (1989) Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
• Sanger F., S. Nicklen, A.R. Coulson (1977) DNA sequencing with chain terminating inhibitors Proc. Natl. Acad. Sci. USA, 74, 5463-5467
• Sweetheart P.J. (1993) Use of peptide libraries to map the substrate specificity of a peptide-modifying enzyme: a 13 consensus peptide stats biotinylation in Escherichia coli Bio / Technology, Oct; 11 (10), 1138-1143
• Sgamarella V., J.H. van de Sande, H.G. Khorana (1979) Studies on the polynucleotides. C. A novel joining reaction catalysed by the T4 polynucleotide ligase Proc. Natl. Acad. Sci. USA, 67, 1468-1475
• Stüber D., H. Matile, G. Garotta (1990) System for high-level production in Escherichia coli and rapid purification of recombinant proteins: Application to epitope mapping, preparation of antibodies and structure-function analysis in: Lefkovits, I., P. Pernis (eds.) Immunological Methods, IV, 121- 152
• Tucker J., Grisshammer (1996) Purification of a rat neurotensin receptor expressed in Escherichia coli Biochem. Journal, 317, 891-899
• Wada K., Wada Y., Ishibashi F., Gojobori T., Ikemura T. (1992) codon usage tabulated from the GenBank genetic sequence data. Nucleic Acid Res., 20, 2111-2118
• Winnacker E.L. (1990) Genes and clones. An introduction to genetic engineering Verlag Chemie, Weinheim, Dearfield Beach, Basel
• Zamenhof P.J., M. Villarejo (1972) Construction and properties of Escherichia coli strains exhibiting α-complementation of β-galactosidase fragments in vivo J. Bacteriol., 110, 171-178 Ritsert et al. (1995): J. Mol. Biol. 235, 151-167

sequence Listings

Claims (50)

Protein conjugate consisting of at least one Function area at any position of the sequence of a Carrier protein range for forming a capsid space structure of the lumazine synthase type, see above that their outer periphery is covalently linked to a plurality of these functional areas. Recombinantly producible protein conjugate consisting from at least one functional protein region at the N-terminus and / or C-terminus and / or inserted into a loop region of the sequence a carrier protein region for forming a capsid space structure of the lumazine synthase type, so that their outer periphery is covalently linked to a variety of functional protein regions. Protein conjugate according to claim 1 or 2, characterized characterized in that the carrier protein region an amino acid sequence, selected from a sequence set, which results from the fact that one for each amino acid position in the sequence of a predetermined native lumazine synthase amino acid or a deletion from the corresponding position of an alignment the predetermined lumazine synthase sequence with at least one native Lumazine sequence of another organism. Protein conjugate according to claim 1 or 2, characterized characterized in that the carrier protein region has the sequence of a native lumazine synthase. Protein conjugate according to claim 1 or 2, characterized characterized in that the carrier protein region has the sequence of a thermostable native lumazine synthase. Protein conjugate according to claim 5, characterized in that that the carrier protein area from a thermostable native lumazine synthase of a hyperthermophilic Microorganism is derived. Protein conjugate according to claim 6, characterized in that that the hyperthermophilic microorganism is Aquifex aeolicus. Protein conjugate according to one of claims 6 or 7, characterized in that the carrier protein region of a Mixed sequence consisting of amino acid positions 1-60 of native lumazine synthase from a mesophilic organism on Bacillus subtilis, and amino acid positions 61-154 of the native lumazine synthase from a hyperthermophilic microorganism, based on Aquifex aeolicus, is constructed. Protein conjugate according to claim 1 or 2, characterized characterized in that the carrier protein region consists of a sequence whose main chain folds into α-helix and β-sheet motifs, wherein 4 β-segments a parallel, central 4-stranded β-sheet form and wherein the central 4-stranded β-sheet on both sides of 2 α-helices each is flanked, so that 5 units of this α-β motifs to a pentameric structure store together, with the N-terminus of each unit in the adjacent unit the fifth β-segment to the central 4-stranded β-sheet where 12 of these pentameric substructures assemble and form an icosahedral capsid structure of a lumazine synthase and the N and C termini of the sequence with those described above Structure features themselves at the surface of the formed icosahedron and wherein the sequence is an alignment of a sequence set different, i. lumazine synthase sequences derived from different organisms, is derivable. Protein conjugate according to claim 1 or 2, characterized characterized in that the carrier protein region is a Sequence of a native lumazine synthase, wherein at least one cysteine unit is replaced or deleted by another amino acid is or is chemically modified. Protein conjugate according to claim 10, characterized in that a cysteine unit at either position 93 and / or position 139 of the lumazine synthase from Bacillus subtilis corresponding position is deleted or exchanged for another amino acid. Protein conjugate according to one of claims 1 to 11, characterized in that the carrier protein region and the functional protein region via a Linker peptide linked together are. Protein conjugate according to one of claims 2 to 12, characterized in that the functional protein region the Sequence of a dihydrofolate reductase, a maltose-binding protein, an in vivo biotinylatable peptide, an antigen-effective Peptides, a surface protein a virus, one recognizable by a monoclonal antibody Peptids, one by chance generated peptide, or is a chemically derivatizable amino acid. Protein conjugate according to one of claims 2 to 13, characterized in that the carrier protein region chemically is modified. Protein conjugate according to one of claims 2 to 14, characterized in that the functional protein region is chemically modified or biotinylated. Heterooligomeric protein conjugate consisting of Mixtures of at least two different protein conjugates according to one of the claims 1 to 15 or at least one protein conjugate according to one of claims 1 to 15 and at least one carrier protein region without functional range with a sequence according to one of claims 3 to 9th Heteropolymeric protein conjugate according to claim 16, characterized in that the proteins of the conjugate by chemical Treatment are covalently linked together. Process for the preparation of a protein conjugate according to claim 1, characterized by the following steps, a) Isolation of a lumazine synthase from a wild-type or a recombinant Organism (carrier protein), b) Chemical coupling of functional molecules to the carrier protein, c) Purification of the protein conjugate. Process for the preparation of a protein conjugate according to one of the claims 2 to 15, characterized by the following steps, a) Preparation a first DNA for the carrier protein area coded, b) fusing at least one second DNA, the for the Functional area with or without linker protein encoded with the 5 'end and / or the 3'-end of the first DNA and / or inserting the second DNA into a loop region of the carrier protein region coding region of the first DNA to form an artificial DNA, c) conversion of the artificial DNA of stage b) into a expression, d) Transformation of host cells with a or more of the expression plasmids obtained in stage c), e) Expression of the artificial DNA in the transformed host cells forming a protein conjugate, f) purification of the protein conjugate, A method according to claim 19, characterized by the introduction a predetermined post-translational modification of the protein conjugate in vivo, between steps e) and f). A method according to claim 19 or 20, characterized by a modification of the protein conjugate by chemical coupling of amino acid residues on the protein surface a capsid spatial structure formed from the protein conjugate with Coupling partners. Method according to one of claims 19 to 21, characterized by a ₁ ) mixing of different protein conjugates obtained according to claim 17 and / or claim 18, b ₁ ) denaturation of the obtained mixture and, c ₁ ) renaturation of the mixture; or by, a ₂ ) denaturing various protein conjugates obtained according to claim 17 and / or claim 18), b ₂ ) mixing the denatured protein conjugates, c ₂ ) renaturing the mixture. Method according to one of claims 19 to 22, characterized to use protein conjugates prepared using a folding support Ligands were prepared. Vector for the preparation of protein conjugates according to one of the claims 2 to 16. DNA for a protein conjugate according to a the claims 2 to 16 coded. Vector or DNA according to claim 24 or 25 by an adapted to the codon usage of Escherichia coli Sequence, which for the lumazine synthase from Aquifex aeolicus encodes. Vector according to Claim 24, characterized that a fused gene for encodes an artificial protein which has a functional protein region and a carrier protein region for forming a capsid space structure of the lumazine synthase type containing or without linker peptide, where the functional protein region with or without linker peptide is itself at the N-terminus of the carrier protein region and wherein the functional DNA portion is at the 5 'end of the carrier protein gene of the lumazine synthase type. Vector according to Claim 27, characterized that he is the gene for the lumazine synthase from Bacillus subtilis, coding for the carrier protein region, the gene for the dihydrofolate reductase from Escherichia coli, coding for the functional protein region and as a linker peptide a DNA fragment coding for contains a tripeptide consisting of the amino acid alanine. Vector according to Claim 27, characterized that he is the gene for the lumazine synthase from Bacillus subtilis, encoding the carrier protein region, the gene for the 'maltose binding Protein 'from Escherichia coli, coding for the functional protein region and as linker peptide a DNA fragment, coding for the amino acid sequence SNNNNNNNNNLGIEGRISEFAAA contains. Vector according to Claim 24, characterized that a fused gene for encodes an artificial protein having a functional protein region and a carrier protein region for forming a capsid space structure of the lumazine synthase type containing or without linker peptide, where the functional protein region with or without linker peptide is itself at the C-terminus of the carrier protein region with the functional DNA portion at the 3 'end of the carrier protein gene of the lumazine synthase type. Vector according to claim 30, characterized in that that he is the gene for the lumazine synthase from Bacillus subtilis, encoding the carrier protein region, the gene for the dihydrofolate reductase from Escherichia coli, coding for the functional protein region and as a linker peptide a DNA fragment coding for the amino acid sequence LAAAGGGG contains. Vector according to Claim 31, characterized that he is the gene for the lumazine synthase from Aquifex aeolicus encoding the carrier protein region and a gene fragment coding for contains the functional protein region with the amino acid sequence GSVDLQPSLIS and via a singular Recognition sequence at the 5 'end the gene sequence of the carrier protein has for the Restriction endonuclease BglII, this site being for fusion from foreign genes to the 5 'end the lumazine synthase is usable. Vector according to claim 24, characterized in that a fused gene codes for an artificial protein which has a functional protein region, a carrier protein region for forming a capsid The lumazine synthase-type structure with or without a linker peptide, wherein the vector contains a DNA fragment coding for a biotinylatable peptide having the sequence LGGIFEAMKMEWR, wherein the amino acid lysine is biotinylatable in vivo. Vector according to claim 33, characterized that he is the gene for the lumazine synthase from Bacillus subtilis, encoding the carrier protein region, and as linker peptide a DNA fragment encoding a tripeptide consisting of the amino acid Alanine contains. Vector according to claim 33, characterized that he adapted to the codon usage of Escherichia coli Gene coding for the lumazine synthase from Aquifex aeolicus as a carrier protein region, and as Linker peptide is a DNA fragment encoding a tripeptide consisting of the amino acid Alanine contains. Vector according to claim 33, characterized that he adapted to the codon usage of Escherichia coli Gene coding for the lumazine synthase from Aquifex aeolicus as a carrier protein region, and as Linker peptide a DNA fragment encoding peptide consisting of the Amino acid sequence HHHHHHAAA contains. Vector according to claim 33, characterized that he adapted to the codon usage of Escherichia coli Gene coding for the lumazine synthase from Aquifex aeolicus as a carrier protein region, and as Linker peptide a DNA fragment encoding peptide consisting of the Amino acid sequence HHHHHHGGSGAAA contains. Vector according to Claim 24, characterized that a fused gene for encodes an artificial protein which has a functional protein region and a lumazine synthase as a carrier protein region contains and wherein the functional protein region is at the N-terminus and wherein the vector contains the following components: a) Lumazine synthase gene from Bacillus subtilis, b) peptide-encoding DNA at the 5 'end of the lumazine synthase gene, the for a foreign peptide of the sequence MGDGAVQPDGGQPAVRNER encoded, wherein the functional DNA portion at the 5 'end of the lumazine synthase gene from Bacillus subtilis and wherein the functional DNA portion for an antigenically active peptide from the VP2 surface protein of the 'Mink enteritis virus' encoded. Vector according to claim 30, characterized in that that is the functional DNA portion at the 3 'end of the lumazine synthase gene from Bacillus subtilis, wherein the functional DNA portion is for an antigenic effective peptide encoded by the VP2 surface protein of the 'Mink enteritis virus' and wherein the fused gene for encodes an artificial protein having a functional protein portion and a carrier protein portion, wherein the functional protein portion at the C-terminus of lumazine synthase and wherein the vector contains the following components: a) Lumazine synthase gene from Bacillus subtilis (without stop codon), b) Peptide-encoding DNA at the 3 'end of the lumazine synthase gene used for a foreign peptide of the sequence GDGAVQPDGGQPAVRNER encoded. Vector according to Claim 24, characterized that is the functional DNA portion at the 5'-end and at the 3 'end of the lumazine synthase gene from Bacillus subtilis and the functional DNA portion for an antigenic effective peptide from the VP2 surface protein of 'Mink enteritis virus' encodes and the fused gene for encodes an artificial protein having a functional protein portion and a carrier protein portion, and wherein the functional protein portion is at both the N-terminus and at the C-terminus of the Lumazine synthase and wherein the vector has the following components includes: a) Lumazine synthase gene from Bacillus subtilis (without stop codon) and b) two peptide coding sequences at the 5 'and the 3' end of the lumazine synthase gene the peptide at the N-terminus, the sequence MGDGAVQPDGGQPAVRNER and the Peptide at the C-terminus, the sequence GDGAVQPDGGQPAVRNER own. Vector according to claim 24, characterized in that the functional DNA region is located at the 5 'end of the lumazine synthase gene from Bacillus subtilis and the functional DNA portion encodes an octapeptide (FLAG peptide) which is recognized by a monoclonal antibody wherein the fused gene encodes an artificial protein containing a functional protein region and a carrier protein region and wherein the functional protein region is located at the N-terminus of the lumazine synthase and wherein the vector contains the following components: a) lumazine synthase gene from Bacillus subtilis, b) peptide-encoding DNA at the 5 'end of the lumazine synthase gene which codes for a foreign peptide of the sequence MDYKDDDDK, and c) DNA coding for a linker peptide with the sequence VKL. Vector according to Claim 24, characterized that is the functional DNA area at the 3 'end of the lumazine synthase gene from Bacillus subtilis, with the functional DNA portion for a hexapeptide (His-6 peptide) which is recognized by a monoclonal antibody, and wherein the fused gene for encodes an artificial protein having a functional protein region and a carrier protein region contains and wherein the functional protein region is at the C-terminus of the lumazine synthase and wherein the vector contains the following components: a) Lumazine synthase gene from Bacillus subtilis without stop codon, b) Peptide-encoding DNA at the 3 'end of the lumazine synthase gene used for a foreign peptide of the sequence HHHHHH encoded. Vector according to Claim 24, characterized that is the functional DNA area at the 3 'end of the lumazine synthase gene from Bacillus subtilis, with the functional DNA region for one encoded artificial peptide sequence associated with the amino acid lysine ends, with the amino acid Lysine for the chemical coupling of functional molecules to the carrier protein region is usable, and wherein the functional protein region as a linker (Tentacle-linker) serves, and wherein the fused gene for an artificial protein which encodes a functional protein region and a carrier protein region contains wherein the functional protein region is located at the C-terminus of the carrier protein region and wherein the vector contains the following components: a) Lumazine synthase gene from Bacillus subtilis, b) codon for lysine at the 3'-end of the artificial DNA, c) DNA coding for a linker peptide having the sequence GGGGSGGGSG. Vector according to Claim 24, characterized that is the functional DNA area at the 3 'end of the lumazine synthase gene from Bacillus subtilis, with the functional DNA region for one encoded artificial peptide sequence with the amino acid cysteine ends, with the amino acid Cysteine for the chemical coupling of functional molecules to the Carrier protein range usable, wherein the functional protein region as a linker (tentacle linker) serves, and wherein the fused gene for an artificial protein which encodes a functional protein region and a carrier protein region contains and wherein the functional protein region is at the C-terminus of the carrier protein region and wherein the vector contains the following components: a) Lumazine synthase gene from Bacillus subtilis (without stop codon), b) Codon for Cysteine at the 3'-end the artificial DNA, c) DNA coding for a linker peptide with the Sequence GGGGSGGGSGGG. Use of protein conjugates according to one of claims 1 to 17 for the preparation of diagnostically or therapeutically usable Antibodies. Use of protein conjugates according to one of claims 1 to 17 for the selective detection of antibodies or for the purification of Antibody mixtures or for the characterization of antibodies. Use of protein conjugates according to one of claims 1 to 17 for the production of protein libraries. Medicaments containing a pharmacologically active Amount of a protein conjugate according to any one of claims 1 to 17th Vaccine containing an immunologically active Amount of a protein conjugate according to one of the claims 1 to 17. Use of protein conjugates according to one of claims 1 to 17 as a biosensor.