WO1991010910A2 - Process for enzymatically treating substrates - Google Patents

Process for enzymatically treating substrates Download PDF

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Publication number
WO1991010910A2
WO1991010910A2 PCT/EP1991/000086 EP9100086W WO9110910A2 WO 1991010910 A2 WO1991010910 A2 WO 1991010910A2 EP 9100086 W EP9100086 W EP 9100086W WO 9110910 A2 WO9110910 A2 WO 9110910A2
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WIPO (PCT)
Prior art keywords
fusion protein
carrier material
binding
binding peptide
peptide
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PCT/EP1991/000086
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German (de)
French (fr)
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WO1991010910A3 (en
Inventor
Rainer Rudolph
Erhard Kopetzki
Stephan Fischer
Adelbert Grossmann
Bärbel HÖLL-NEUGEBAUER
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Boehringer Mannheim Gmbh
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Priority claimed from DE4001508A external-priority patent/DE4001508A1/en
Priority claimed from DE4002636A external-priority patent/DE4002636A1/en
Application filed by Boehringer Mannheim Gmbh filed Critical Boehringer Mannheim Gmbh
Publication of WO1991010910A2 publication Critical patent/WO1991010910A2/en
Priority to FI914389A priority Critical patent/FI914389A0/en
Priority to KR1019910701147A priority patent/KR920701822A/en
Priority to NO91913673A priority patent/NO913673L/en
Publication of WO1991010910A3 publication Critical patent/WO1991010910A3/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G01N33/56988—HIV or HTLV
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/735—Fusion polypeptide containing domain for protein-protein interaction containing a domain for self-assembly, e.g. a viral coat protein (includes phage display)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16211—Human Immunodeficiency Virus, HIV concerning HIV gagpol
    • C12N2740/16222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the invention relates to a method for the enzymatic treatment of substrates.
  • enzymes are widely used in biotechnology and diagnostics. In biotechnology, enzymes are used to specifically convert substrates. For example, Starch derivatives can be prepared by treatment with amylase. In diagnostics, specific reactions are carried out with enzymes, which lead to an evaluable signal. Furthermore, it is sometimes necessary for diagnostics to specifically convert substrates before they are detected. For example, cholesterol esters in the sample must be saponified beforehand to determine cholesterol. For many of these applications it is advantageous to immobilize enzymes. This is usually done by binding enzymes to a carrier material. The enzymes are usually bound covalently via spacers which are bound to functional groups of the enzyme.
  • a problem with this is that the binding must take place in such a way that the enzymatic properties of the enzyme are not impaired by the fixation, and furthermore that the binding takes place in such a way that the active center remains accessible to the substrates.
  • the binding of the enzyme should be such that there is no detachment under the conditions during the reaction with the substrate.
  • the enzymes used for the above-mentioned purposes are often produced by genetic engineering.
  • a gene coding for the desired enzyme is inserted into a plasmid, transformed into a suitable organism and expressed.
  • the enzymes are obtained from the lysate after cell disruption.
  • the cleaning of the enzyme often causes difficulties, since after cell disruption in the lysate obtained a great deal many proteins are present, the separation of which is absolutely necessary for further use.
  • a method for the enzymatic treatment of substrates which is characterized in that the substrate to be treated is brought into contact with a biocatalyst which has been obtained by using a gene which codes for a biologically active substance and a DNA Fragment which codes for a binding peptide which can interact with a carrier material, combined to produce a fusion protein, inserted into a suitable vector, cultured after transformation into a suitable organism, which unlocks the cells, and the lysate containing the fusion protein brings into contact a carrier material which is capable of binding to the binding peptide, the fusion protein binding to the carrier material by means of intermolecular interaction and then recovering the enzymatically treated substrate.
  • the enzyme in the form of a fusion protein is immobilized on a support material in a non-covalent bond by intermolecular interaction in such a way that the bond is not dissolved under the conditions of the substrate conversion, so that the immobilized enzyme is available for further reaction cycles.
  • binding via the peptide sequence fused to the N- or C-terminal end of the enzyme achieved several advantages.
  • the binding does not affect the active center and the mobility of the enzyme. This improves the accessibility of the substrate to the immobilized enzyme.
  • purification is not necessary since the enzyme can be bound to the carrier material directly from the lysate via the binding peptide.
  • the immobilized enzyme for regeneration of the biocatalyst can be detached from the carrier material in a manner known to the person skilled in the art and the latter can be loaded again with enzyme-containing lysate without the emptying of the bioreactor vessel or the disposal of the carrier material being necessary for this.
  • a biocatalyst is used for the enzymatic treatment of substrates, which contains a carrier material to which a fusion protein is bound via intermolecular interactions.
  • This fusion protein consists of the enzyme required for the process and a binding peptide mediating the binding to the carrier material. All enzymes that can be produced by genetic engineering, such as e.g.
  • ⁇ -glucosidase glucose oxidase, aminoacylase, glucose isomerase, creatinase, ⁇ -galactosidase, pullulanase, trehalase, trehalose phosphorylase, glucose dehydrogenase, mannitol dehydrogenase, D-amino acid oxidase (D-AOD), aldolase, cholesterol dehydrogenase (alcohol esterase) ADH), pig liver esterase, subtilisin, dehalogenases, naphthalene dioxygenase, chymotrypsin, ß-amylase and thermostable amylases, ligninase, nitrile hydratase, horseradish peroxidase.
  • Amino acid sequences which can be produced by genetic engineering and which can interact with a carrier material are suitable as the binding peptide.
  • the fusion protein can be produced by known molecular biological methods (T. Man
  • the enzyme and binding peptide coding DNA fragments inserted into a suitable vector.
  • the vector is then transformed into a suitable organism and after selection this organism is then cultivated in the usual way.
  • the cultured cells are disrupted by known methods and the lysate obtained in the disruption, which contains the fusion protein, is brought into contact with the carrier material.
  • the fusion protein binds to the carrier material via the binding peptide and can thus be separated from the other substances contained in the lysate.
  • a material that can enter into an intermolecular interaction with the binding peptide is used as the carrier material.
  • Intermolecular interactions that are suitable for fixing the fusion protein to the carrier material are ionic, hydrophilic interactions, complex formation, hydrophobic interactions, binding peptide / receptor interactions and signal peptide / membrane interactions.
  • the carrier material and sequence of the binding peptide are therefore selected so that they can enter into one of these interactions. Proteins and carrier materials suitable for this are known to the person skilled in the art.
  • the binding preferably takes place via ionic interaction, with both the carrier material and the binding peptide of the fusion protein having charged groups or via the formation of complexes such as those e.g. is used in metal chelate affinity chromatography.
  • the carrier materials used are above all gels, such as those used for chromatography or ion exchange resins.
  • Mechanically resilient supports are particularly suitable, in particular hydrophilic polymers referred to as fracture gels and matrices referred to as tentacle gels, which carry exchange centers on tentacle-like polymer chains which are bound to a hydrophilic matrix.
  • Others such as the so-called soft gels and known for chromatography, based on polysaccharides, materia Lines such as dextrins, agarose or sepharose are suitable in a correspondingly derivatized form.
  • As derivatives for example
  • Fractogel® EMD SO 3 --650 is preferably used as the carrier material if the binding peptide contains arginine and lysine as amino acids. When using glutamic acid and aspartic acid for the binding peptide, the use of Fractogel® EMD TMAE-650 is preferred.
  • chelating resins such as TSK chelates 5-PW or chelating Sepharose® 6B ff, which have iminodiacetate as ligands and tris (carboxymethyl) ethylenediamine agarose, are suitable for binding the fusion proteins.
  • a column material which contains the metal chelator nitrilotriacetic acid (NTA) as the binding to the fusion protein-mediating ligand is particularly preferably used as the carrier material.
  • NTA metal chelator nitrilotriacetic acid
  • the protein sequence of the fusion protein responsible for the binding then has poly-histidine. The binding then takes place via carrier-bound metal ions by complex formation with the functional histidine residues.
  • a carrier material which has negatively or positively charged groups is particularly preferably used.
  • the protein sequence of the fusion protein responsible for the binding then has oppositely charged groups which can be introduced by the amino acids lysine and / or arginine or glutamic acid and / or aspartic acid. The
  • the binding peptide part of the fusion protein consists of several amino acids, the length of the peptide being selected so that the fusion protein is sufficiently strongly adhered to the carrier material and the active sites of the enzyme are accessible for binding the substrate.
  • the binding peptide preferably consists of 2 to 30 amino acids.
  • the binding peptide not only consists of the amino acids which have the functional groups required for binding to the carrier material, but also contains several amino acids which improve the accessibility of the enzyme.
  • the introduction of a proline or glycine polymer with 1 to 10 amino acid units is particularly suitable.
  • the amino acid sequence for the binding protein is suitably selected so that it is largely resistant to proteinase digestion.
  • the binding peptide does not have to consist of a homogeneous chain of the same amino acids. It can also have a combination of similarly charged amino acids.
  • the binding peptide preferably consists of a sequence which contains the amino acids arginine and / or lysine or aspartic acid and / or glutamic acid.
  • the free end of the binding peptide is preferably protected by a fused amino acid which is not charged and is difficult to access for exoproteinases, such as proline.
  • a fused amino acid which is not charged and is difficult to access for exoproteinases, such as proline.
  • the enzyme contained in the fusion protein is present in partially denatured form due to the workup and in this case cannot develop its full activity, in a preferred embodiment the enzyme is renatured after fixation on the carrier according to methods known per se, first treatment with a denaturing agent Agent followed by a renaturation step. The conditions are set so that the fusion protein cannot be detached.
  • a particular advantage here is that the reaggregation that would otherwise be feared during renaturation is not possible due to the fixation.
  • the lysate obtained after cell disruption is brought into contact with the carrier material under conditions which favor the binding.
  • the binding peptide binds via its functional groups to the corresponding functional groups of the carrier material.
  • the biologically active immobilized enzyme thus obtained can then be used for the treatment of substrates.
  • the substrate is brought into contact with the immobilized enzyme in a conventional manner and the enzymatically treated substrate is then obtained.
  • the immobilized enzyme used according to the invention is preferably filled into a column and the substrate is passed over it in a manner known per se.
  • a further object of the invention was to provide solid phase-bound or particle-bound, specifically bindable peptide and protein substances (receptors) for use in enzyme immunoassays, in which the receptors are bound in a simple manner in such a way that their immunological activity is not impaired and that there is no detachment under the reaction conditions when the immunoassay is carried out.
  • This object is achieved by a method for the detection of specifically bindable substances according to the principle of immunoassay using a solid phase-bound receptor, which is characterized in that the sample solution and at least one labeled receptor which is bindable with the substance to be detected are brought into contact with a solid-phase bound receptor obtained by combining a gene coding for an immunologically active substance and a DNA fragment coding for a binding peptide which can interact with a carrier material to produce a fusion protein, inserted into a suitable vector, transformed into a suitable organism, cultivating the organism, disrupting the cells and bringing the lysate containing the fusion protein into contact with a carrier material which is capable of binding to the binding peptide, the fusion protein being bound to the carrier material by intermolecular changes binding and separates the solid from the liquid phase after incubation and determines the marking in one of the two phases.
  • a method for the detection of specifically bindable substances according to the principle of the homogeneous immunoassay using a particle-bound receptor is provided, which is characterized in that the sample is at least one with the substance to be detected bindable particle-bound receptor incubated, which was obtained by inserting a gene coding for an immunologically active substance and combining a DNA fragment coding for a binding peptide which can interact with a carrier material to produce a fusion protein, into a suitable vector , transformed into a suitable organism, cultivating the organism, disrupting the cells and bringing the lysate containing the fusion protein into contact with a carrier material which is capable of binding to the binding peptide, the fusion protein being attached to the carrier material by intermoles specific interaction binds and determines the agglutination after incubation.
  • the solid-phase-bound or particle-bound receptor used is immobilized in the form of a fusion protein by intermolecular interaction on a carrier material in a non-covalent bond, the carrier material being able to be present either as a solid phase, for example in the form of tubes, microtiter plates or polystyrene balls, or in Form of particles, eg latex particles.
  • the fusion protein consists of the immunologically active substance required for the method and a binding peptide mediating the binding to the carrier material.
  • All immunologically active substances that can be produced by genetic engineering are suitable for the method according to the invention, for example antibodies and their fragments and antigens, for example HIV or hepatitis antigens or haptens such as hormones, medicaments etc.
  • the same peptides as described above are suitable as binding peptides were.
  • a material which is suitable as a solid phase or particles for use in an immunoassay and which can enter into an intermolecular interaction with the binding peptide is used as the carrier material. Support materials suitable for this are known to the person skilled in the art.
  • the method for producing the fusion protein is as described above.
  • the solid phase-bound or particle-bound receptors thus produced can be used in a manner known per se for immunoassays of the heterogeneous and homogeneous type.
  • the execution of these immunoassays is known to the person skilled in the art and requires no further explanation here.
  • the sample solution is implemented with at least two receptors, with one receptor being solid-phase bound in the case of the heterogeneous immunoassay and the other being marked, and one receptor being particle-bound in the case of the homogeneous immunoassay and the other being bound with the substance to be detected and can enter the particle-bound receptor.
  • Complexes form from the Receptors and the substance to be detected, which cause a signal change, in the case of heterogeneous immunoassays by the label, which can be an enzyme, a fluorescent, chemiluminescent or radioactive substance, and in the case of homogeneous immunoassays by agglutination.
  • the label which can be an enzyme, a fluorescent, chemiluminescent or radioactive substance, and in the case of homogeneous immunoassays by agglutination.
  • Figure 1 shows the construction of the ⁇ -glucosidase-Arg6 expression vector
  • Figure 2a shows the plasmid map of pKKl77-3 / GLUCPI. ARG6;
  • FIG. 3 shows a diagram of a maltose biocatalyst with complete substrate conversion
  • Substrate buffer 10 mM KPP, 1 mM EDTA,
  • Substrate buffer 10 mM KPP, 1 mM EDTA, 0.15 M maltose, pH 7.0;
  • Denaturation buffer 10 mM KPP, 1 mM EDTA,
  • Renaturation buffer 10 mM KPP, 1 mM EDTA,
  • E. coli strain RM82 a methionine revertant from ED8654, (Murray et al. 1977, Mol. Gen. Genet. 150, 53-61), which (i) the R plasmid pREM6677
  • the structural gene of the ⁇ -glucosidase PI from baker's yeast was extended at the 3 'end by a DNA fragment which codes for the amino acid sequence GlyArgArgArgArgArgArg. This results in an ⁇ -glucosidase-PI fusion protein which contains 6 additional arginine residues (polycationic anchor sequence) and glycine as a "spacer" at the C-terminal.
  • the approximately 4.7 kbp plasmid pKK177-3 / GLUCPI (preparation and description see: EP-A 0 300 425, Kopetzki, Schumacher, Buckel, 1989, Mol. Gen. Genet. 216, 149-155) was partial with the restriction endonuclease EcoRI and completely digested with Bcll. The synthetic DNA fragment was inserted into the approximately 4.7 kbp long Bell / EcoRI vector fragment
  • the plasmid pKKl77-3 / EH became about 130 bp long
  • the E. coli strain RM82 For the expression of the ⁇ -glucosidase-PI-Arg6 gene, the E. coli strain RM82 was used, which has the ⁇ -glucosidase-PI-ARG6 expression plasmid and an R-plasmid, which is used for the lacI q repressor and for a trimethoprim - Resistance coded. Expression of the ⁇ -glucosidase is under the control of the tac hybrid promoter.
  • 500 ml modified M9 minimal medium (6 g Na 2 HPO 4 / l, 3 g KH 2 PO 4 / l, 0.5 g NaCl / l, 1 g NH 4 Cl / l, 2.5 mg thiamine / l, 5 G Casamino Acids / l, 20 ml glycerol / l (87%), 0.25 g MgSO 4 ⁇ 7 H 2 O / l, 50 mg ampicillin / l, 10 mg trimethoprim / 1) were mixed with 1% of an overnight culture of E. coli strain RM82-I q / pKK177-3 / GLUCPI. ARG6 (also in the above medium) inoculated. The cultures were incubated at 30 ° C with constant shaking. The growth was monitored by measuring the optical density at 550 ⁇ m (OD 550nm ).
  • the increase in the ⁇ -glucosidase content in the cells was monitored using 1 ml culture samples taken and analyzed at various times after induction.
  • the cell pellet from 1 ml of E. coli culture was resuspended in 0.5 ml of 10 mM phosphate buffer, 1 mM EDTA, pH 7.0, the cells were disrupted by ultrasound, the cell debris was separated off by centrifugation and the supernatant was further processed as cell lysate.
  • KPP potassium phosphate buffer
  • 1 EDTA 0.068 atm
  • the cation exchanger Fraktogel® EMD SO 3 - - 650 (“tentacle gel”) was used, whose exchanger groups are freely movable in the room on flexible polymer chains. This ensures a good interaction of the column material with the proteins to be bound.
  • the cation exchanger Fractogel® EMD SO 3 - -650 was equilibrated with 10 mM potassium phosphate buffer, 1 mM EDTA pH 7.0.
  • Heparin-Sepharose® CL-6B was used as the gel matrix.
  • the binding capacity for the ⁇ -glucosidase-Arg6 fusion protein was determined to be 3000 U / ml for both gel materials.
  • the crude extract ( ⁇ -glucosidase activity approx. 200 U / ml) was pumped at a speed of approx. 10 column volumes / Hour applied to the column. Then the column was 10 mM Potassium phosphate buffer, 1 mM EDTA pH 7.0 and the loading of the column material with the ⁇ -glucosidase Arg6
  • Fusion protein determined. For this purpose, the total activity of ⁇ -glucosidase not bound to the column was measured in the column eluate (flow and washing buffer) and subtracted from the amount of enzyme applied to the column. The binding of the ⁇ -glucosidase-Arg6 fusion protein to Fractogel® SO 3 - -650 or
  • Heparin-Sepharose® CL-6B was 95-98%.
  • the enzymatic hydrolysis properties compared to the ⁇ -glucosidase substrates maltose and p-NPG were determined.
  • the glucose released from the maltose by the ⁇ -glucosidase catalyst per unit of time or the nitrophenol released from p-NPG is a measure of the catalyst performance.
  • the release of p-nitrophenol from p-NPG was determined photometrically in the column eluate (analogous to ⁇ -glucosidase determination).
  • the hydrolysis of maltose in the column eluate was followed on the basis of the glucose released.
  • E 2 is read at 366 nm.
  • the glucose content of the sample was calculated from the difference E 2 - E 1 .
  • Purified ⁇ -glucosidase-Arg6 fusion protein (approx. 80 U / mg) was denatured in 10 mM potassium phosphate buffer, 1 mM EDTA, 6 M urea, 2 mM DTE pH 7.0 and applied to a "tentacle gel" column equilibrated with denaturing buffer. After sample application, the column was rinsed with renaturation buffer (10 mM potassium phosphate (KPP), 1 mM EDTA, 2 mM 1,4-dithioerythritol (DTE), pH 7.0) (approx. 10 column volumes).
  • KPP potassium phosphate
  • DTE 1,4-dithioerythritol
  • the bound and renatured ⁇ -glucosidase-Arg6 fusion protein was eluted with 10 mM potassium phosphate buffer, 1 mM EDTA, 1 M NaCl, pH 7.0. In the eluate obtained with 1 M NaCl, a specific ⁇ -glucosidase activity of approx.
  • the carrier material Fractogel® EMD SO 3 --650 was loaded in a batch process with purified ⁇ -glucosidase-Arg6 fusion protein (approx. 800 U), filled into a column, for one hour with denaturing buffer (10 mM KPP, 1 mM EDTA, 2 mM DTE, 6M Urea, pH 6.8) and then washed with substrate buffer (10 mM KPP, 1 mM EDTA, 0.15 M maltose, pH 6.8).
  • HIV polypeptide HIV2 (envgp32) -HIVl (polp32-envgp41-gagpl7-p24-15-poly (Arg-Lys) is a "multifunctional" HIV fusion polypeptide produced by means of recombinant DNA technology. It consists of protein segments of the gag, pol and env region of the HIVl and the env region of the HIV2 retrovirus.
  • the HIV polypeptide C-terminal has a polycationic anchor sequence of 13 positively charged amino acids (arginine and lysine).
  • the protein sequence of the HIV polypeptide is shown in SEQ ID NO: 2.
  • DSM 5446 was used (a lactose revertant of RM82), which contains the HIV fusion protein expression plasmid pKK233-2 / MYYL_gp32_polp32_gp41_p24-polyArgLys (ampicillin resistance) and the dnaY-lacI q plasmid pUBS500.
  • the construction of the HIV expressin plasmid is described in German patent application P 40 02 636.1 dated January 30, 1990.
  • pUBS500 (EP-A 0 373 365) is a pACYC derivative (Chang and Cohen, J. Bacteriol. 134 (1978) 1141-1156) which, in addition to kanamycin resistance, has a lacl q repressor gene (Carlos, Nature 274 (1978) 762 -765) and a gene for t-RNA arginine (Anticodone: AAG, AGG), which is rare in E. coli.
  • the recombinant E. coli cells were grown at 30 ° C. in DYT medium (16 g bactotrypton, 10 g yeast extract, 5 g NaCl per liter), supplemented with 50 mg / 1 ampicillin plus 50 mg / l kanamycin. After reaching an optical density of 0.6-0.8 at 550 nm, the cells were induced with IPTG (isopropyl- ⁇ -D-thiogalactopyranoside, final concentration 1 mM). After an induction time of 4 to 10 hours, the cells were centrifuged off, washed with 10 mM Tris-HCl buffer, pH 7.0 and stored at -20 ° C. until further processing.
  • DYT medium 16 g bactotrypton, 10 g yeast extract, 5 g NaCl per liter
  • IPTG isopropyl- ⁇ -D-thiogalactopyranoside, final concentration 1 mM
  • E. coli cells 20 g (wet weight) of E. coli cells were resuspended in 400 ml of 100 mM Tris-HCl buffer, pH 6.5-7.5, 0.25 mg / ml of lysozyme were added and incubated for 0.5-1 hour at room temperature. The suspension was then cooled to 0-4 ° C. and the cells contained therein were disrupted by ultrasonic treatment (French Press). The cell debris and the insoluble, aggregated HIV fusion protein ("inclusion bodies”) were separated by centrifugation and the pellet was washed 1-2 times with 200 -400 ml of 0.5 M NaCl or KCl and 1% (v / v) Triton-X-100.
  • the pellet was in Resuspended 20 ml of 50 mM Tris-HCl pH 8.0 with 8 M urea and 5 mM ⁇ -mercaptoethanol with stirring (magnetic stirrer) at room temperature for 1 hour and the insoluble cell components were removed by centrifugation.
  • the protein concentration of the solubilized proteins was modified according to Bradford (Anal. Biochem. 72 (1976) 248-254) according to Gotham et al. (Anal. Biochem. 173 (1988) 353-358).
  • the sample (10-100 ul human serum) was diluted hundreds of times in PBS with 10% calf serum and incubated for 4-12 hours at 37 ° C with 10 ul gel coated with HIV fusion protein. The mixture was then washed three times with 1-1.5 ml of washing solution (0.5% Tween 20 in demineralized water).
  • a conjugate of peroxidase and polyclonal antibody (POD conjugate, approx. 30 mU peroxidase / ml in PBS), which is directed against the Fc ⁇ part of human IgG, was incubated for 4 to 12 hours and three times with washing solution washed.
  • POD conjugate approx. 30 mU peroxidase / ml in PBS
  • the culture is kept for at least 5 years after receipt of the last application for a sample.

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Abstract

In order to treat a substrate with enzymes, the substrate to be treated is brought into contact with a biocatalyzer obtained by combining a gene that codes for a biologically active substance and a fragment of DNA that codes for a binding peptide capable of interacting with a supporting base, to produce a fusion protein. The fusion protein is inserted in an appropriate vector, transformed in an appropriate organism, the organism is cultivated, its cells are dissolved and the lysate containing the fusion protein is brought into contact with a supporting base capable of binding with the binding peptide. The fusion protein is bound to the supporting base by intermolecular interaction, and the enzymatically treated substrate is thus finally obtained.

Description

Verfahren zur enzymatischen Behandlung von Substraten  Process for the enzymatic treatment of substrates
BES CHRE I BUNG DESCRIPTION
Die Erfindung betrifft ein Verfahren zur enzymatischen Behandlung von Substraten.  The invention relates to a method for the enzymatic treatment of substrates.
Aufgrund ihrer Substratspezifität werden Enzyme in großem Umfang in der Biotechnologie und in der Diagnostik eingesetzt. In der Biotechnologie werden Enzyme verwendet, um Substrate gezielt umzuwandeln. So können z.B. Stärkederivate durch Behandlung mit Amylase hergestellt werden. In der Diagnostik werden mit Enzymen spezifische Umsetzungen durchgeführt, die zu einem auswertbaren Signal führen. Weiterhin ist es für die Diagnostik teilweise erforderlich, Substrate vor ihrem Nachweis spezifisch umzuwandeln. So müssen beispielsweise für die Cholesterinbestimmung in der Probe vorhandene Cholesterinester vorher verseift werden. Für viele dieser Anwendungen ist es vorteilhaft, Enzyme zu immobilisieren. In der Regel geschieht dies, indem Enzyme an ein Trägermaterial gebunden werden. Die Bindung der Enzyme erfolgt dabei üblicherweise kovalent über Spacer, die an funktioneile Gruppen des Enzyms gebunden werden. Ein Problem dabei ist, daß die Bindung so erfolgen muß, daß die enzymatischen Eigenschaften des Enzyms durch die Fixierung nicht beeinträchtigt werden, und weiterhin, daß die Bindung so erfolgt, daß das aktive Zentrum für die Substrate zugänglich bleibt. Außerdem soll die Bindung des Enzyms so sein, daß bei den Bedingungen während der Umsetzung mit dem Substrat keine Ablösung erfolgt. Due to their substrate specificity, enzymes are widely used in biotechnology and diagnostics. In biotechnology, enzymes are used to specifically convert substrates. For example, Starch derivatives can be prepared by treatment with amylase. In diagnostics, specific reactions are carried out with enzymes, which lead to an evaluable signal. Furthermore, it is sometimes necessary for diagnostics to specifically convert substrates before they are detected. For example, cholesterol esters in the sample must be saponified beforehand to determine cholesterol. For many of these applications it is advantageous to immobilize enzymes. This is usually done by binding enzymes to a carrier material. The enzymes are usually bound covalently via spacers which are bound to functional groups of the enzyme. A problem with this is that the binding must take place in such a way that the enzymatic properties of the enzyme are not impaired by the fixation, and furthermore that the binding takes place in such a way that the active center remains accessible to the substrates. In addition, the binding of the enzyme should be such that there is no detachment under the conditions during the reaction with the substrate.
Die für die obengenannten Zwecke verwendeten Enzyme werden häufig auf gentechnologischem Wege hergestellt. Dazu wird ein das gewünschte Enzym kodierende Gen in ein Plasmid insertiert, in einen geeigneten Organismus transformiert und exprimiert. Die Enzyme werden nach Zellaufschluß aus dem Lysat gewonnen. Schwierigkeiten macht häufig die Reinigung des Enzyms, da nach Zellaufschluß in dem erhaltenen Lysat sehr viele Proteine vorliegen, deren Abtrennung für die weitere Verwendung unbedingt erforderlich ist. The enzymes used for the above-mentioned purposes are often produced by genetic engineering. For this purpose, a gene coding for the desired enzyme is inserted into a plasmid, transformed into a suitable organism and expressed. The enzymes are obtained from the lysate after cell disruption. The cleaning of the enzyme often causes difficulties, since after cell disruption in the lysate obtained a great deal many proteins are present, the separation of which is absolutely necessary for further use.
Es war daher Aufgabe der Erfindung, für die enzymatische Behandlung von Substraten einen Biokatalysator zur Verfügung zu stellen, in dem die Enzyme in reiner Form auf einfache Weise so gebunden werden, daß ihre Aktivität nicht beeinträchtigt wird und daß eine Ablösung unter den Reaktionsbedingungen bei der Gewinnung des behandelten Substrats nicht erfolgt, so daß der Biokatalysator für möglichst viele Umsatzzyklen zur Verfügung steht. It was therefore an object of the invention to provide a biocatalyst for the enzymatic treatment of substrates in which the enzymes are bound in a simple manner in a simple manner so that their activity is not impaired and that detachment under the reaction conditions during the extraction of the treated substrate is not carried out, so that the biocatalyst is available for as many sales cycles as possible.
Diese Aufgabe wird gelöst durch ein Verfahren zur enzymatischen Behandlung von Substraten, das dadurch gekennzeichnet ist, daß man das zu behandelnde Substrat mit einem Biokatalysator in Kontakt bringt, der erhalten wurde, indem man ein Gen, das für eine biologisch aktive Substanz kodiert und ein DNA-Fragment, das für ein Bindepeptid, das mit einem Trägermaterial in Wechselwirkung treten kann, kodiert, zur Erzeugung eines Fusionsproteins kombiniert, in einen geeigneten Vektor insertiert, nach Transformation in einen geeigneten Organismus kultiviert, die Zellen aufschließt, und das das Fusionsprotein enthaltende Lysat mit einem Trägermaterial in Kontakt bringt, das mit dem Bindepeptid bindefähig ist, wobei das Fusionsprotein an das Trägermaterial durch zwischenmolekulare Wechselwirkung bindet und anschließend das enzymatisch behandelte Substrat gewinnt. This object is achieved by a method for the enzymatic treatment of substrates, which is characterized in that the substrate to be treated is brought into contact with a biocatalyst which has been obtained by using a gene which codes for a biologically active substance and a DNA Fragment which codes for a binding peptide which can interact with a carrier material, combined to produce a fusion protein, inserted into a suitable vector, cultured after transformation into a suitable organism, which unlocks the cells, and the lysate containing the fusion protein brings into contact a carrier material which is capable of binding to the binding peptide, the fusion protein binding to the carrier material by means of intermolecular interaction and then recovering the enzymatically treated substrate.
Erfindungsgemäß wird zur Erzeugung des Biokatalysators das Enzym in Form eines Fusionsproteins durch zwischenmolekulare Wechselwirkung an ein Trägermaterial in nicht kovalenter Bindung so immobilisiert, daß die Bindung unter den Bedingungen der Substratumsetzung nicht gelöst wird, wodurch das immobilisierte Enzym für weitere Reaktionszyklen zur Verfügung steht. Durch die Bindung über die am N- oder C-terminalen Ende des Enzyms anfusionierte Peptidsequenz werden mehrere Vorteile erzielt. Die Bindung beeinträchtigt nicht das aktive Zentrum und die Beweglichkeit des Enzyms. Dadurch wird die Zugänglichkeit für das Substrat zum immobilisierten Enzym verbessert. Darüberhinaus ist eine Aufreinigung nicht erforderlich, da das Enzym über das Bindepeptid direkt aus dem Lysat an das Trägermaterial gebunden werden kann. According to the invention, in order to produce the biocatalyst, the enzyme in the form of a fusion protein is immobilized on a support material in a non-covalent bond by intermolecular interaction in such a way that the bond is not dissolved under the conditions of the substrate conversion, so that the immobilized enzyme is available for further reaction cycles. By binding via the peptide sequence fused to the N- or C-terminal end of the enzyme achieved several advantages. The binding does not affect the active center and the mobility of the enzyme. This improves the accessibility of the substrate to the immobilized enzyme. In addition, purification is not necessary since the enzyme can be bound to the carrier material directly from the lysate via the binding peptide.
Weiterhin kann das immobilisierte Enzym zur Regenerierung des Biokatalysators in dem Fachmann bekannter Weise vom Trägermaterial abgelöst und letzteres erneut mit Enzym-haltigem Lysat beladen werden, ohne daß hierzu die Entleerung des Bioreaktorgefäßes oder die Entsorgung des Trägermaterials erforderlich ist. Furthermore, the immobilized enzyme for regeneration of the biocatalyst can be detached from the carrier material in a manner known to the person skilled in the art and the latter can be loaded again with enzyme-containing lysate without the emptying of the bioreactor vessel or the disposal of the carrier material being necessary for this.
Erfindungsgemäß wird zur enzymatischen Behandlung von Substraten ein Biokatalysator verwendet, der ein Trägermaterial enthält, an das über zwischenmolekulare Wechselwirkungen ein Fusionsprotein gebunden ist. Dieses Fusionsprotein besteht aus dem für das Verfahren benötigten Enzym und einem die Bindung an das Trägermaterial vermittelnden Bindepeptid. Für das erfindungsgemäße Verfahren sind alle Enzyme geeignet, die gentechnologisch hergestellt werden können wie z.B. α-Glucosidase, Glucoseoxidase, Aminoacylase, Glucose-Isomerase, Creatinase, ß-Galactosidase, Pullulanase, Trehalase, Trehalose-Phosphorylase, Glucose Dehydrogenase, Mannitol-Dehydrogenase, D-Aminosäureoxidase (D-AOD), Aldolase, Cholesterin- esterase, Alkoholdehydrogenase (ADH), Schweineleberesterase, Subtilisin, Dehalogenasen, Naphthalin-Dioxygenase, Chymotrypsin, ß-Amylase und thermostabile Amylasen, Ligninase, Nitrilhydratase, Meerrettich-Peroxidase. Als Bindepeptid sind gentechnologisch herstellbare Aminosäuresequenzen geeignet, die mit einem Trägermaterial eine Wechselwirkung eingehen können. Das Fusionsprotein kann nach bekannten molekularbiologischen Verfahren hergestellt werden (T. Maniatis, E.F. According to the invention, a biocatalyst is used for the enzymatic treatment of substrates, which contains a carrier material to which a fusion protein is bound via intermolecular interactions. This fusion protein consists of the enzyme required for the process and a binding peptide mediating the binding to the carrier material. All enzymes that can be produced by genetic engineering, such as e.g. α-glucosidase, glucose oxidase, aminoacylase, glucose isomerase, creatinase, β-galactosidase, pullulanase, trehalase, trehalose phosphorylase, glucose dehydrogenase, mannitol dehydrogenase, D-amino acid oxidase (D-AOD), aldolase, cholesterol dehydrogenase (alcohol esterase) ADH), pig liver esterase, subtilisin, dehalogenases, naphthalene dioxygenase, chymotrypsin, ß-amylase and thermostable amylases, ligninase, nitrile hydratase, horseradish peroxidase. Amino acid sequences which can be produced by genetic engineering and which can interact with a carrier material are suitable as the binding peptide. The fusion protein can be produced by known molecular biological methods (T. Maniatis, E.F.
Fritsch und Sambrook, J. Molecular Cloning, 1982, Cold Spring Harbor Laboratory). Dazu werden die Enzym und Bindepeptid kodierenden DNA-Fragmente in einen geeigneten Vektor insertiert. Der Vektor wird dann in einen geeigneten Organismus transformiert und nach Selektion wird dieser Organismus dann in üblicher Weise kultiviert. Die gezüchteten Zellen werden nach bekannten Verfahren aufgeschlossen und das beim Aufschluß erhaltene Lysat, das das Fusionsprotein enthält, mit dem Trägermaterial in Kontakt gebracht. Über das Bindepeptid bindet das Fusionsprotein an das Trägermaterial und kann so von den anderen im Lysat enthaltenen Substanzen abgetrennt werden. Fritsch and Sambrook, J. Molecular Cloning, 1982, Cold Spring Harbor Laboratory). To do this, the enzyme and binding peptide coding DNA fragments inserted into a suitable vector. The vector is then transformed into a suitable organism and after selection this organism is then cultivated in the usual way. The cultured cells are disrupted by known methods and the lysate obtained in the disruption, which contains the fusion protein, is brought into contact with the carrier material. The fusion protein binds to the carrier material via the binding peptide and can thus be separated from the other substances contained in the lysate.
Als Trägermaterial wird ein Material verwendet, das mit dem Bindepeptid eine zwischenmolekulare Wechselwirkung eingehen kann. Zwischenmolekulare Wechselwirkungen, die für die Fixierung des Fusionsproteins am Trägermaterial geeignet sind, sind ionische, hydrophile Wechselwirkungen, Komplexbildung, hydrophobe Wechselwirkungen, Bindepeptid/Rezeptorwechselwirkungen sowie Signalpeptid/Membran-Wechselwirkungen. Trägermaterial und Sequenz des Bindepeptids werden daher so ausgewählt, daß sie eine dieser Wechselwirkungen eingehen können. Hierfür geeignete Proteine und Trägermaterialien sind dem Fachmann bekannt. Bevorzugt erfolgt die Bindung über ionische Wechselwirkung, wobei sowohl Trägermaterial als auch Bindepeptid des Fusionsproteins geladene Gruppen aufweisen oder über die Bildung von Komplexen, wie sie z.B. bei der Metallchelat-Affinitätschromatographie ausgenutzt wird. A material that can enter into an intermolecular interaction with the binding peptide is used as the carrier material. Intermolecular interactions that are suitable for fixing the fusion protein to the carrier material are ionic, hydrophilic interactions, complex formation, hydrophobic interactions, binding peptide / receptor interactions and signal peptide / membrane interactions. The carrier material and sequence of the binding peptide are therefore selected so that they can enter into one of these interactions. Proteins and carrier materials suitable for this are known to the person skilled in the art. The binding preferably takes place via ionic interaction, with both the carrier material and the binding peptide of the fusion protein having charged groups or via the formation of complexes such as those e.g. is used in metal chelate affinity chromatography.
Als Trägermaterialien kommen vor allem Gele, wie sie auch für die Chromatographie eingesetzt werden oder Ionenaustauschharze zur Anwendung. Besonders geeignet sind mechanisch belastbare Träger, insbesondere als Fraktogele bezeichnete hydrophile Polymerisate und als Tentakelgele bezeichnete Matrices, die an tentakelartigen Polymerketten, die an eine hydrophile Matrix gebunden sind, Austauscherzentren tragen. Auch andere, wie z.B. die als Softgele bezeichneten und für die Chromatographie bekannten, auf Polysacchariden basierenden Materia lien wie Dextrine, Agarose oder Sepharose sind in entsprechend derivatisierter Form geeignet. Als Derivate können beispielsweise The carrier materials used are above all gels, such as those used for chromatography or ion exchange resins. Mechanically resilient supports are particularly suitable, in particular hydrophilic polymers referred to as fracture gels and matrices referred to as tentacle gels, which carry exchange centers on tentacle-like polymer chains which are bound to a hydrophilic matrix. Others, such as the so-called soft gels and known for chromatography, based on polysaccharides, materia Lines such as dextrins, agarose or sepharose are suitable in a correspondingly derivatized form. As derivatives, for example
S-Sepharose® ff (Pharmacia/LKB)  S-Sepharose® ff (Pharmacia / LKB)
CM-Sepharose® ff (Pharmacia/LKB)  CM-Sepharose® ff (Pharmacia / LKB)
SP-Sephadex® C-50 (Pharmacia/LKB), für batch-Anwendung  SP-Sephadex® C-50 (Pharmacia / LKB), for batch application
SE-EUPERGIT® (Röhm Pharma)  SE-EUPERGIT® (Röhm Pharma)
Bio-Rex® 70 (Bio-Rad)  Bio-Rex® 70 (Bio-Rad)
Heparin-Sepharose® CL-6B  Heparin-Sepharose® CL-6B
Fractogel® TSK SP-650 (Toyo Soda/Merck)  Fractogel® TSK SP-650 (Toyo Soda / Merck)
Fractogel® EMD SO3- -650 (Merck, "Tentakelgel") Fractogel® EMD SO 3 - -650 (Merck, "Tentacle Gel")
Q-Sepharose® ff (Pharmacia/LKB)  Q-Sepharose® ff (Pharmacia / LKB)
DEAE-Sepharose® ff (Pharmacia/LKB)  DEAE-Sepharose® ff (Pharmacia / LKB)
DEAE-Sephadex® A-50 (Pharmacia/LKB), für batch-Anwendung QAM- EUPERGIT® (Röhm Pharma)  DEAE-Sephadex® A-50 (Pharmacia / LKB), for batch application QAM-EUPERGIT® (Röhm Pharma)
AG MP-1 (Bio-Rad) AG MP-1 (Bio-Rad)
Fractogel® TSK DEAE-650 (Toyo Soda/Merck)  Fractogel® TSK DEAE-650 (Toyo Soda / Merck)
Fractogel® EMD TMAE-650 (Merck, "Tentakelgel") Fractogel® EMD TMAE-650 (Merck, "Tentacle Gel")
verwendet werden. Bevorzugt wird als Trägermaterial Fractogel® EMD SO3--650 eingesetzt, wenn das Bindepeptid Arginin und Lysin als Aminosäuren enthält. Bei Verwendung von Glutaminsäure und Asparaginsäure für das Bindepeptid ist der Einsatz von Fractogel® EMD TMAE-650 bevorzugt. be used. Fractogel® EMD SO 3 --650 is preferably used as the carrier material if the binding peptide contains arginine and lysine as amino acids. When using glutamic acid and aspartic acid for the binding peptide, the use of Fractogel® EMD TMAE-650 is preferred.
Weiterhin sind chelatbildende Harze wie z.B. TSK-Chelate 5-PW oder Chelating Sepharose® 6B ff, die Iminodiacetat als Liganden aufweisen sowie tris-(carboxymethyl)-ethylendiamin-Agarose für die Bindung der Fusionsproteine geeignet. Besonders bevorzugt wird als Trägermaterial ein Säulenmaterial verwendet, das den Metallchelator Nitrilotriessigsäure (NTA) als die Bindung zum Fusionsprotein vermittelnden Liganden enthält. Die für die Bindung verantwortliche Proteinsequenz des Fusionsproteins weist dann poly-Histidin auf. Die Bindung erfolgt dann über trägergebundene Metallionen durch Komplexbildung mit den funktioneilen Histidin-Resten. Besonders bevorzugt wird ein Trägermaterial verwendet, das negativ bzw. positiv geladene Gruppen aufweist. Die für die Bindung verantwortliche Proteinsequenz des Fusionsproteins weist dann entgegengesetzt geladene Gruppen auf, die durch die Aminosäuren Lysin und/oder Arginin bzw. Glutaminsäure und/oder Asparaginsäure eingeführt werden können. Die Bindung erfolgt dann über polyionische Wechselwirkungen. Furthermore, chelating resins such as TSK chelates 5-PW or chelating Sepharose® 6B ff, which have iminodiacetate as ligands and tris (carboxymethyl) ethylenediamine agarose, are suitable for binding the fusion proteins. A column material which contains the metal chelator nitrilotriacetic acid (NTA) as the binding to the fusion protein-mediating ligand is particularly preferably used as the carrier material. The protein sequence of the fusion protein responsible for the binding then has poly-histidine. The binding then takes place via carrier-bound metal ions by complex formation with the functional histidine residues. A carrier material which has negatively or positively charged groups is particularly preferably used. The protein sequence of the fusion protein responsible for the binding then has oppositely charged groups which can be introduced by the amino acids lysine and / or arginine or glutamic acid and / or aspartic acid. The binding then takes place via polyionic interactions.
Der Bindepeptidteil des Fusionsproteins besteht aus mehreren Aminosäuren, wobei die Länge des Peptids so ausgewählt wird, daß eine ausreichend starke Haftung des Fusionsproteins am Trägermaterial gewährleistet ist und die aktiven Stellen des Enzyms für die Bindung des Substrats zugänglich sind. Bevorzugt besteht das Bindepeptid aus 2 bis 30 Aminosäuren. In einer bevorzugten Ausführungsform besteht das Bindepeptid nicht nur aus den Aminosäuren, die die für die Bindung an das Trägermaterial erforderlichen funktioneilen Gruppen aufweisen, sondern enthält darüberhinaus noch mehrere Aminosäuren, die die Zugänglichkeit des Enzyms verbessern. Besonders geeignet ist die Einführung eines Prolin- oder Glycinpolymers mit 1 bis 10 Aminosäureeinheiten. Die Aminosäuresequenz für das Bindungsprotein wird geeigneterweise so ausgewählt, daß sie- gegen Proteinaseverdau weitgehend resistent ist. The binding peptide part of the fusion protein consists of several amino acids, the length of the peptide being selected so that the fusion protein is sufficiently strongly adhered to the carrier material and the active sites of the enzyme are accessible for binding the substrate. The binding peptide preferably consists of 2 to 30 amino acids. In a preferred embodiment, the binding peptide not only consists of the amino acids which have the functional groups required for binding to the carrier material, but also contains several amino acids which improve the accessibility of the enzyme. The introduction of a proline or glycine polymer with 1 to 10 amino acid units is particularly suitable. The amino acid sequence for the binding protein is suitably selected so that it is largely resistant to proteinase digestion.
Das Bindepeptid muß nicht aus einer homogenen Kette gleicher Aminosäuren bestehen. Es kann ebenso eine Kombination gleichartig geladener Aminosäuren aufweisen. Bevorzugt besteht das Bindepeptid aus einer Sequenz, die die Aminosäuren Arginin und/oder Lysin bzw. Asparaginsäure und/oder Glutaminsäure enthält. The binding peptide does not have to consist of a homogeneous chain of the same amino acids. It can also have a combination of similarly charged amino acids. The binding peptide preferably consists of a sequence which contains the amino acids arginine and / or lysine or aspartic acid and / or glutamic acid.
Um den Bindepeptidanteil des Fusionsproteins gegen einen proteolytischen Verdau zu schützen, wird bevorzugt das freie Ende des Bindepeptids durch eine anfusionierte Aminosäure, die nicht geladen ist und für Exoproteinasen schwer zugänglich ist, wie z.B. Prolin, geschützt. Da das im Fusionsprotein enthaltene Enzym durch die Aufarbeitung in teilweise denaturierter Form vorliegen und in diesem Fall nicht seine volle Aktivität entfalten kann, wird in einer bevorzugten Ausführungsform das Enzym nach Fixierung am Träger renaturiert nach an sich bekannten Methoden, wobei zuerst eine Behandlung mit einem denaturierenden Agens und anschließend ein Renaturierungsschritt erfolgt. Die Bedingungen werden dabei so eingestellt, daß keine Ablösung des Fusionsproteins erfolgen kann. Besonderer Vorteil hierbei ist, daß die bei einer Renaturierung ansonsten zu befürchtende Reaggregation nicht möglich ist aufgrund der Fixierung. In order to protect the binding peptide portion of the fusion protein against proteolytic digestion, the free end of the binding peptide is preferably protected by a fused amino acid which is not charged and is difficult to access for exoproteinases, such as proline. Since the enzyme contained in the fusion protein is present in partially denatured form due to the workup and in this case cannot develop its full activity, in a preferred embodiment the enzyme is renatured after fixation on the carrier according to methods known per se, first treatment with a denaturing agent Agent followed by a renaturation step. The conditions are set so that the fusion protein cannot be detached. A particular advantage here is that the reaggregation that would otherwise be feared during renaturation is not possible due to the fixation.
Zur Bindung des Fusionsproteins an das Trägermaterial wird das nach Zellaufschluß erhaltene Lysat mit dem Trägermaterial unter die Bindung begünstigenden Bedingungen in Kontakt gebracht. Dabei bindet das Bindepeptid über seine funktioneilen Gruppen an die entsprechenden funktionellen Gruppen des Trägermaterials. Das so erhaltene, biologisch aktive immobilisierte Enzym kann dann zur Behandlung von Substraten eingesetzt werden. In üblicher Weise wird das Substrat mit dem immobilisierten Enzym in Kontakt gebracht und anschließend das enzymatisch behandelte Substrat gewonnen. Bevorzugt wird das erfindungsgemäß verwendete immobilisierte Enzym in eine Säule gefüllt und das Substrat in an sich bekannter Weise darübergeleitet. To bind the fusion protein to the carrier material, the lysate obtained after cell disruption is brought into contact with the carrier material under conditions which favor the binding. The binding peptide binds via its functional groups to the corresponding functional groups of the carrier material. The biologically active immobilized enzyme thus obtained can then be used for the treatment of substrates. The substrate is brought into contact with the immobilized enzyme in a conventional manner and the enzymatically treated substrate is then obtained. The immobilized enzyme used according to the invention is preferably filled into a column and the substrate is passed over it in a manner known per se.
Eine weitere Aufgabe der Erfindung war es, Festphasen-gebundene bzw. Partikel-gebundene, spezifisch bindefähige Peptid- und Proteinsubstanzen (Rezeptoren) zur Verwendung in EnzymImmunoassays zur Verfügung zu stellen, bei denen die Rezeptoren in reiner Form auf einfache Weise so gebunden werden, daß ihre immunologische Aktivität nicht beeinträchtigt wird und daß eine Ablösung unter den Reaktionsbedingungen bei der Durchführung des Immunoassays nicht erfolgt. Diese Aufgabe wird gelöst durch ein Verfahren zum Nachweis von spezifisch bindefähigen Substanzen nach dem Prinzip des Immunoassays unter Verwendung eines Festphasen-gebundenen Rezeptors, das dadurch gekennzeichnet ist, daß man die Probelösung sowie mindestens einen markierten, mit der nachzuweisenden Substanz bindefähigen Rezeptor in Kontakt bringt mit einem Festphasen-gebundenen Rezeptor, der erhalten wurde, indem man ein Gen, das für eine immunologisch aktive Substanz kodiert und ein DNA-Fragment, das für ein Bindepeptid, das mit einem Trägermaterial in Wechselwirkung treten kann, kodiert, zur Erzeugung eines Fusionsproteins kombiniert, in einen geeigneten Vektor insertiert, in einen geeigneten Organismus transformiert, den Organismus kultiviert, die Zellen aufschließt und das das Fusionsprotein enthaltende Lysat mit einem Trägermaterial in Kontakt bringt, das mit dem Bindepeptid bindefähig ist, wobei das Fusionsprotein an das Trägermaterial durch zwischenmolekulare Wechselwirkung bindet und nach Inkubation die feste von der flüssigen Phase trennt und die Markierung in einer der beiden Phasen bestimmt. A further object of the invention was to provide solid phase-bound or particle-bound, specifically bindable peptide and protein substances (receptors) for use in enzyme immunoassays, in which the receptors are bound in a simple manner in such a way that their immunological activity is not impaired and that there is no detachment under the reaction conditions when the immunoassay is carried out. This object is achieved by a method for the detection of specifically bindable substances according to the principle of immunoassay using a solid phase-bound receptor, which is characterized in that the sample solution and at least one labeled receptor which is bindable with the substance to be detected are brought into contact with a solid-phase bound receptor obtained by combining a gene coding for an immunologically active substance and a DNA fragment coding for a binding peptide which can interact with a carrier material to produce a fusion protein, inserted into a suitable vector, transformed into a suitable organism, cultivating the organism, disrupting the cells and bringing the lysate containing the fusion protein into contact with a carrier material which is capable of binding to the binding peptide, the fusion protein being bound to the carrier material by intermolecular changes binding and separates the solid from the liquid phase after incubation and determines the marking in one of the two phases.
Als weitere Ausführungsform wird ein Verfahren zum Nachweis von spezifisch bindefähigen Substanzen nach dem Prinzip des homogenen Immunoassays unter Verwendung eines Partikel-gebundenen Rezeptors zur Verfügung gestellt, das dadurch gekennzeichnet ist, daß man die Probe mindestens mit einem mit der nachzuweisenden Substanz bindefähigen Partikel-gebundenen Rezeptor inkubiert, der erhalten wurde, indem man ein Gen das für eine immunologisch aktive Substanz kodiert und ein DNA- Fragment, das für ein Bindepeptid, das mit einem Trägermaterial in Wechselwirkung treten kann, kodiert, zur Erzeugung eines Fusionsproteins kombiniert, in einen geeigneten Vektor insertiert, in einen geeigneten Organismus transformiert, den Organismus kultiviert, die Zellen aufschließt und das das Fusionsprotein enthaltende Lysat mit einem Trägermaterial in Kontakt bringt, das mit dem Bindepeptid bindefähig ist, wobei das Fusionsprotein an das Trägermaterial durch zwischenmole kulare Wechselwirkung bindet und nach Inkubation die Agglutination bestimmt. As a further embodiment, a method for the detection of specifically bindable substances according to the principle of the homogeneous immunoassay using a particle-bound receptor is provided, which is characterized in that the sample is at least one with the substance to be detected bindable particle-bound receptor incubated, which was obtained by inserting a gene coding for an immunologically active substance and combining a DNA fragment coding for a binding peptide which can interact with a carrier material to produce a fusion protein, into a suitable vector , transformed into a suitable organism, cultivating the organism, disrupting the cells and bringing the lysate containing the fusion protein into contact with a carrier material which is capable of binding to the binding peptide, the fusion protein being attached to the carrier material by intermoles specific interaction binds and determines the agglutination after incubation.
Erfindungsgemäß wird der verwendete Festphasen-gebundene bzw. Partikel-gebundene Rezeptor in Form eines Fusionsproteins durch zwischenmolekulare Wechselwirkung an ein Trägermaterial in nicht kovalenter Bindung immobilisiert, wobei das Trägermaterial entweder als feste Phase, z.B. in Form von Röhrchen, Mikrotiterplatten oder Polystyrolkugeln vorliegen kann oder in Form von Partikeln, z.B. Latexteilchen. Das Fusionsprotein besteht aus der für das Verfahren benötigten, immunologisch aktiven Substanz und einem die Bindung an das Trägermaterial vermittelnden Bindepeptid. Für das erfindungsgemäße Verfahren sind alle immunologisch aktiven Substanzen geeignet, die gentechnologisch hergestellt werden können, z.B. Antikörper und deren Fragmente sowie Antigene, z.B. HIV oder Hepatitis- Antigene oder Haptene wie Hormone, Arzneimittel etc. Als Bindepeptid sind dieselben Peptide geeignet, wie sie vorher beschrieben wurden. Als Trägermaterial wird ein Material verwendet, das als Festphase bzw. Partikel zur Verwendung in einem Immunoassay geeignet ist und mit dem Bindepeptid eine zwischenmolekulare Wechselwirkung eingehen kann. Hierfür geeignete Trägermaterialien sind dem Fachmann bekannt. Das Verfahren zur Herstellung des Fusionsproteins erfolgt wie oben beschrieben. Die so hergestellten Festphasen-gebundenen bzw. Partikel-gebundenen Rezeptoren können in an sich bekannter Weise für Immunoassays der heterogenen und homogenen Art eingesetzt werden. Die Durchführung dieser Immunoassays ist dem Fachmann bekannt und bedarf hier keiner näheren Erläuterung. Prinzipiell wird die Probelösung dabei mit mindestens zwei Rezeptoren umgesetzt, wobei im Fall des heterogenen Immunoassays ein Rezeptor Festphasen-gebunden ist und der andere markiert ist und im Fall des homogenen Immunoassays ein Rezeptor Partikel-gebunden ist und der andere eine Bindung mit der nachzuweisenden Substanz und dem Partikel-gebundenen Rezeptor eingehen kann. Es bilden sich Komplexe aus den Rezeptoren und der nachzuweisenden Substanz, die eine Signaländerung bewirken, im Fall des heterogenen Immunoassays durch die Markierung, die ein Enzym, eine fluoreszierende, chemilumineszierende oder radioaktive Substanz sein kann und im Falle des homogenen Immunoassays durch Agglutination. According to the invention, the solid-phase-bound or particle-bound receptor used is immobilized in the form of a fusion protein by intermolecular interaction on a carrier material in a non-covalent bond, the carrier material being able to be present either as a solid phase, for example in the form of tubes, microtiter plates or polystyrene balls, or in Form of particles, eg latex particles. The fusion protein consists of the immunologically active substance required for the method and a binding peptide mediating the binding to the carrier material. All immunologically active substances that can be produced by genetic engineering are suitable for the method according to the invention, for example antibodies and their fragments and antigens, for example HIV or hepatitis antigens or haptens such as hormones, medicaments etc. The same peptides as described above are suitable as binding peptides were. A material which is suitable as a solid phase or particles for use in an immunoassay and which can enter into an intermolecular interaction with the binding peptide is used as the carrier material. Support materials suitable for this are known to the person skilled in the art. The method for producing the fusion protein is as described above. The solid phase-bound or particle-bound receptors thus produced can be used in a manner known per se for immunoassays of the heterogeneous and homogeneous type. The execution of these immunoassays is known to the person skilled in the art and requires no further explanation here. In principle, the sample solution is implemented with at least two receptors, with one receptor being solid-phase bound in the case of the heterogeneous immunoassay and the other being marked, and one receptor being particle-bound in the case of the homogeneous immunoassay and the other being bound with the substance to be detected and can enter the particle-bound receptor. Complexes form from the Receptors and the substance to be detected, which cause a signal change, in the case of heterogeneous immunoassays by the label, which can be an enzyme, a fluorescent, chemiluminescent or radioactive substance, and in the case of homogeneous immunoassays by agglutination.
Die Erfindung wird durch die folgenden Figuren und Beispiele erläutert: The invention is illustrated by the following figures and examples:
Fig. 1 zeigt die Konstruktion des α-Glucosidase-Arg6 Expressionsvektors; Figure 1 shows the construction of the α-glucosidase-Arg6 expression vector;
Fig. 2a zeigt die Plasmidkarte von pKKl77-3/GLUCPI. ARG6; Figure 2a shows the plasmid map of pKKl77-3 / GLUCPI. ARG6;
Fig. 2b zeigt die Nukleotid-Sequenz des Plasmids 2b shows the nucleotide sequence of the plasmid
PKK177-3/GLUCPI. ARG6;  PKK177-3 / GLUCPI. ARG6;
Fig. 3 zeigt ein Diagramm eines Maltose-Biokatalysators mit vollständigem Substratumsatz; 3 shows a diagram of a maltose biocatalyst with complete substrate conversion;
Substratpuffer: 10 mM KPP, 1 mM EDTA,  Substrate buffer: 10 mM KPP, 1 mM EDTA,
6 mM Maltose, pH 7,0;  6 mM maltose, pH 7.0;
Fig. 4 zeigt ein Diagramm eines Maltose-Biokatalysators mit Substratsättigung limitiertem Substratumsatz Substratpuffer: 10 mM KPP, 1 mM EDTA, 0,15 M Maltose, pH 7,0; 4 shows a diagram of a maltose biocatalyst with substrate saturation limited substrate conversion. Substrate buffer: 10 mM KPP, 1 mM EDTA, 0.15 M maltose, pH 7.0;
Fig. 5 zeigt die Renaturierungskinetik von am Träger denaturierter α-Glucosidase Arg6 5 shows the renaturation kinetics of α-glucosidase Arg6 denatured on the support
Denaturierungspuffer: 10 mM KPP, 1 mM EDTA,  Denaturation buffer: 10 mM KPP, 1 mM EDTA,
2 mM DTE, 6 M Harnstoff, pH 6,8  2mM DTE, 6M urea, pH 6.8
Renaturierungspuffer: 10 mM KPP, 1 mM EDTA,  Renaturation buffer: 10 mM KPP, 1 mM EDTA,
0,15 M Maltose, pH 6,8. B e i s p i e l e 0.15 M maltose, pH 6.8. Examples
Zur Manipulation von DNA wurden Standardmethoden benutzt, wie sie bei Maniatis et al. (1982) in Molecular Cloning, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, beschrieben sind. Die verwendeten molekularbiologischen Reagenzien wurden nach den Angaben des Herstellers eingesetzt. Standard methods were used to manipulate DNA, as described in Maniatis et al. (1982) in Molecular Cloning, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724. The molecular biological reagents used were used according to the manufacturer's instructions.
Materialien: Materials:
Restriktionsendonukleasen und T4-DNA-Ligase waren von Boehringer Mannheim GmbH, Hefeextrakt, Bacto Trypton und Casamino Acids waren von Difco.  Restriction endonucleases and T4 DNA ligase were from Boehringer Mannheim GmbH, yeast extract, Bacto Trypton and Casamino Acids were from Difco.
Bakterienstämme: Bacterial strains:
E. coli-Stamm HB101 (Maniatis et al., 1982, Molecular  E. coli strain HB101 (Maniatis et al., 1982, Molecular
cloning: A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York) oder GM48 (Yanisch- Perron et al., 1985, Gene 3.3, 103-119) für die Präparation von unmethylierter DNA, wurden als Rezipientenstämme für die Plasmidamplifikation benutzt. Der E. coli-Stamm RM82, eine Methionin Revertante von ED8654, (Murray et al. 1977, Mol. Gen. Genet. 150, 53-61), der (i) das R-Plasmid pREM6677 cloning: A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York) or GM48 (Yanisch-Perron et al., 1985, Gene 3.3, 103-119) for the preparation of unmethylated DNA were used as recipient strains for plasmid amplification. E. coli strain RM82, a methionine revertant from ED8654, (Murray et al. 1977, Mol. Gen. Genet. 150, 53-61), which (i) the R plasmid pREM6677
(pePAH9, DSM 3691P), das für den laclq-Repressor und für die Trimethoprim-Resistenz codiert, und (ii) das α-Glucosidase PI-Arg6 Expressionsplasmid pKKl77-3/GLUC ARG6 (Ampicillin Resistenz) enthält, wurde für die Expression des α-Glucosidase-PI-Arg6-Fusionsproteins verwendet. Dieser Stamm wird im folgenden mit RM82 Iq/pKK177-3/GLUCPI_ARG6 bezeichnet. (pePAH9, DSM 3691P) coding for the lacl q repressor and for trimethoprim resistance, and (ii) containing the α-glucosidase PI-Arg6 expression plasmid pKKl77-3 / GLUC ARG6 (ampicillin resistance) was used for expression of the α-glucosidase-PI-Arg6 fusion protein. This strain is hereinafter referred to as RM82 I q / pKK177-3 / GLUCPI_ARG6.
B e i s p i e l 1 Example 1
Konstruktion des α-Glucosidase-Arg6 Expressionsplasmids pKK177-3/GLUCPI-ARG6 Construction of the α-glucosidase-Arg6 expression plasmid pKK177-3 / GLUCPI-ARG6
Das Strukturgen der α-Glucosidase PI aus Bäckerhefe wurde am 3'-Ende um ein DNA-Fragment erweitert, das für die Aminosäuresequenz GlyArgArgArgArgArgArg kodiert. Dadurch entsteht ein α-Glucosidase-PI-Fusionsprotein, das C-terminal 6 zusätzliche Argininreste (polykationische Ankersequenz) und Glycin als "Spacer" enthält. The structural gene of the α-glucosidase PI from baker's yeast was extended at the 3 'end by a DNA fragment which codes for the amino acid sequence GlyArgArgArgArgArgArg. This results in an α-glucosidase-PI fusion protein which contains 6 additional arginine residues (polycationic anchor sequence) and glycine as a "spacer" at the C-terminal.
Hierzu wurde das ca. 4,7 kBp lange Plasmid pKK177-3/GLUCPI (Herstellung und Beschreibung siehe: EP-A 0 300 425, Kopetzki, Schumacher, Buckel, 1989, Mol. Gen. Genet. 216, 149-155) partiell mit der Restriktionsendonuklease EcoRI und vollständig mit Bcll verdaut. In das isolierte ca. 4,7 kBp lange Bell/EcoRI-Vektorfragment wurde das synthetische DNA-Fragment For this purpose the approximately 4.7 kbp plasmid pKK177-3 / GLUCPI (preparation and description see: EP-A 0 300 425, Kopetzki, Schumacher, Buckel, 1989, Mol. Gen. Genet. 216, 149-155) was partial with the restriction endonuclease EcoRI and completely digested with Bcll. The synthetic DNA fragment was inserted into the approximately 4.7 kbp long Bell / EcoRI vector fragment
5'-AATTATGACGATATCC-3' 5'-AATTATGACGATATCC-3 '
3'-TACTGCTATAGGCTAG-5'  3'-TACTGCTATAGGCTAG-5 '
MetThrlleSer.... ligiert (Konstruktion: pKK177-3/GLUCPI. SD), wodurch die EcoRI und Bell Restriktionsschnittstelle zerstört werden. Aus dem Plasmid YRp/GLUCPI (DSM 4173P) (Herstellung und Beschreibung siehe EP-A 0 323 838; Kopetzki, Buckel, Schumacher, 1989, Yeast 5, 11-24) wurde das ca. 300 Bp lange EcoRI-Fragment isoliert. Dieses wurde mit Hinfl nachgespalten und das ca. 90 Bp lange EcoRI/HinfI-Fragment isoliert.  MetThrlleSer .... ligated (construction: pKK177-3 / GLUCPI. SD), whereby the EcoRI and Bell restriction interface are destroyed. The approximately 300 bp EcoRI fragment was isolated from the plasmid YRp / GLUCPI (DSM 4173P) (for preparation and description see EP-A 0 323 838; Kopetzki, Buckel, Schumacher, 1989, Yeast 5, 11-24). This was cleaved with Hinfl and the approximately 90 bp EcoRI / HinfI fragment was isolated.
Danach wurde das ca. 90 Bp lange EcoRI/HinfI-Fragment, sowie das synthetische DNA-Fragment IleTyrLeuValLysGlyArgArgArgArgArgArgEndEnd 5'-AATCTACCTGGTCAAAGGGCGCCGACGTCGCCGGCGTTAATA Then the approximately 90 bp EcoRI / HinfI fragment, as well as the synthetic DNA fragment IleTyrLeuValLysGlyArgArgArgArgArgArgEndEnd 5'-AATCTACCTGGTCAAAGGGCGCCGACGTCGCCGGCGTTAATA
3'-GATGGACCAGTTTCCCGCGGCTGCAGCGGCCGCAATTATTCGA-5 ' ------- ------ 3'-GATGGACCAGTTTCCCGCGGCTGCAGCGGCCGCAATTATTCGA-5 '------- ------
Hinfl Hindlll über eine Dreifachligation in das ca. 2.85 kBp lange Hindfll via a triple ligation into the approx. 2.85 kBp long
EcoRI/HindIII-pKKl77-3-Vektorfragment (DSM 3062) insertiert (Konstruktion: pKK177-3/EH).  EcoRI / HindIII pKKl77-3 vector fragment (DSM 3062) inserted (construction: pKK177-3 / EH).
Aus dem Plasmid pKKl77-3/EH wurde das ca. 130 Bp lange The plasmid pKKl77-3 / EH became about 130 bp long
EcoRI/HindiII-Fragment isoliert und in das ca. 4.65 kBp lange EcoRI/HindIII-pKK177-3/GLUCPI-SD Vektorfragment ligiert (Konstruktion: pKKl77-3/GLUCPI. ARG6). Durch Restriktionsanalyse und DNA-Sequenzierung wurde der korrekte Zusammenbau des α- Glucosidase-Arg6 Gens bestätigt. Die Konstruktion des α-Glu- cosidase-Arg6 Expressionsvektors ist schematisch in Abbildung 1 dargestellt. Zur Expression des α-Glucosidase-Arg6 Fusionsproteins wurde das Plasmid pKK177-3/GLUCPI ARG6 (Abbildung 2a und 2b, SEQ ID NO:1) in den E.coli Stamm RM82. Iq (ED. lq , DSM 2102) transformiert. EcoRI / HindiII fragment isolated and ligated into the approximately 4.65 kbp long EcoRI / HindIII-pKK177-3 / GLUCPI-SD vector fragment (construction: pKKl77-3 / GLUCPI. ARG6). The correct assembly of the α-glucosidase-Arg6 gene was confirmed by restriction analysis and DNA sequencing. The construction of the α-glucosidase-Arg6 expression vector is shown schematically in Figure 1. To express the α-glucosidase-Arg6 fusion protein, the plasmid pKK177-3 / GLUCPI ARG6 (Figures 2a and 2b, SEQ ID NO: 1) was inserted into the E.coli strain RM82. I q (ED. L q , DSM 2102) transformed.
B e i s p i e l 2 Example: 2
Expression des α-Glucosidase-Arg6 Fusionsproteins in E.coli Expression of the α-glucosidase-Arg6 fusion protein in E. coli
Für die Expression des α-Glucosidase-PI-Arg6 Gens wurde der E.coli Stamm RM82 verwendet, der das α-Glucosidase-PI-ARG6 Expressionsplasmid und ein R-Plasmid besitzt, das für den lacIq-Repressor, sowie für eine Trimethoprim-Resistenz codiert. Die Expression der α-Glucosidase steht unter Kontrolle des tac-Hybridpromotors. For the expression of the α-glucosidase-PI-Arg6 gene, the E. coli strain RM82 was used, which has the α-glucosidase-PI-ARG6 expression plasmid and an R-plasmid, which is used for the lacI q repressor and for a trimethoprim - Resistance coded. Expression of the α-glucosidase is under the control of the tac hybrid promoter.
Kulturbedingungen Cultural conditions
500 ml modifiziertes M9-Minimalmedium (6 g Na2HPO4/l, 3 g KH2PO4/l, 0,5 g NaCl/l, 1 g NH4Cl/l, 2,5 mg Thiamin/l, 5 g Casamino Acids/l, 20 ml Glycerin/l (87%), 0,25 g MgSO4 · 7 H2O/l, 50 mg Ampicillin/l, 10 mg Trimethoprim/1) wurden mit 1 % einer Übernachtkultur des E. coli Stammes RM82-Iq /pKK177- 3/GLUCPI. ARG6 (ebenfalls in o. g. Medium) inoculiert. Die Kulturen wurden bei 30°C unter konstantem Schütteln inkubiert. Das Wachstum wurde durch Messung der optischen Dichte bei 550 um verfolgt (OD550nm). 500 ml modified M9 minimal medium (6 g Na 2 HPO 4 / l, 3 g KH 2 PO 4 / l, 0.5 g NaCl / l, 1 g NH 4 Cl / l, 2.5 mg thiamine / l, 5 G Casamino Acids / l, 20 ml glycerol / l (87%), 0.25 g MgSO 4 · 7 H 2 O / l, 50 mg ampicillin / l, 10 mg trimethoprim / 1) were mixed with 1% of an overnight culture of E. coli strain RM82-I q / pKK177-3 / GLUCPI. ARG6 (also in the above medium) inoculated. The cultures were incubated at 30 ° C with constant shaking. The growth was monitored by measuring the optical density at 550 µm (OD 550nm ).
Induktion der α-Glucosidase Induction of α-glucosidase
Nach Erreichen einer Zelldichte von OD550nm 0,7 - 0,9 wurde durch Zugabe von Lactose (Endkonzentration 0,5 %) die Bildung der α-Glucosidase induziert. Nach ca. 16 Stunden wurden die Zellen durch Zentrifugation geerntet. After reaching a cell density of OD 550 nm 0.7-0.9, the formation of the α-glucosidase was induced by adding lactose (final concentration 0.5%). After approximately 16 hours, the cells were harvested by centrifugation.
Der Anstieg des α-Glucosidase-Gehalts in den Zellen wurde anhand von 1 ml -Kulturproben verfolgt, die zu verschiedenen Zeiten nach der Induktion entnommen und analysiert wurden. The increase in the α-glucosidase content in the cells was monitored using 1 ml culture samples taken and analyzed at various times after induction.
Bestimmung der α-Glucosidase-Aktivität Determination of α-glucosidase activity
Das Zellpellet aus 1 ml E. coli Kultur wurde in 0,5 ml 10 mM Phosphatpuffer, 1 mM EDTA, pH 7,0 resuspendiert, die Zellen mittels Ultraschall aufgeschlossen, die Zelltrümmer durch Zentrifugation abgetrennt und der Überstand als Zellysat weiterverarbeitet. The cell pellet from 1 ml of E. coli culture was resuspended in 0.5 ml of 10 mM phosphate buffer, 1 mM EDTA, pH 7.0, the cells were disrupted by ultrasound, the cell debris was separated off by centrifugation and the supernatant was further processed as cell lysate.
50 μl des Überstandes (evtl. verdünnen) wurden mit 3 ml 50 μl of the supernatant (possibly diluted) were mixed with 3 ml
Kaliumphosphat-Puffer (100 mM, pH 6,8), der das α-Glucosidase-Substrat p-Nitrophenyl-α-D-glucopyranosid (p-NPG; 2 mM) enthielt, vermischt. Die α-Glucosidase setzte durch enzymatische Spaltung p-Nitrophenol frei. Dies wurde anhand der Extinktionszunahme bei 405 nm bestimmt. B e i s p i e l 3 Potassium phosphate buffer (100 mM, pH 6.8) containing the α-glucosidase substrate p-nitrophenyl-α-D-glucopyranoside (p-NPG; 2 mM) was mixed. The α-glucosidase released p-nitrophenol by enzymatic cleavage. This was determined on the basis of the increase in extinction at 405 nm. Example 3
Herstellung des Biokatalysators Production of the biocatalyst
Zellaufschluß und Gewinnung des Rohextrakts Cell disruption and extraction of the crude extract
Das Zellpellet aus 500 ml E. coli-Kultur wurde in 30 - 50 ml 10 mM Kaliumphosphat-Puffer (KPP), 1 mM EDTA, pH 7,0 resuspendiert, mechanisch mittels French Press (2 Durchgänge bei 14000 psi; 1 psi = 0,068 atm) aufgeschlossen und anschließend die Zeiltrümmerfragmente durch Zentrifugation abgetrennt (10 min, 7000 rpm). Das so gewonnene Zell-Lysat wurde direkt zur Herstellung des Biokatalysators verwendet. The cell pellet from 500 ml of E. coli culture was resuspended in 30-50 ml of 10 mM potassium phosphate buffer (KPP), 1 mM EDTA, pH 7.0, mechanically using a French press (2 passes at 14000 psi; 1 psi = 0.068 atm) and then the debris fragments separated by centrifugation (10 min, 7000 rpm). The cell lysate obtained in this way was used directly to produce the biocatalyst.
Trägermaterialien Carrier materials
Verwendung fand der Kationenaustauscher Fraktogel® EMD SO3- - 650 ("Tentakelgel"), dessen Austauschergruppen an flexiblen Polymerketten frei beweglich im Raum stehen. Dadurch wird eine gute Wechselwirkung des Säulenmaterials mit den zu bindenden Proteinen gewährleistet. The cation exchanger Fraktogel® EMD SO 3 - - 650 ("tentacle gel") was used, whose exchanger groups are freely movable in the room on flexible polymer chains. This ensures a good interaction of the column material with the proteins to be bound.
Der Kationenaustauscher Fractogel® EMD SO3- -650 wurde mit 10 mM Kaliumphosphat-Puffer, 1 mM EDTA pH 7,0 äquilibriert. Alternativ wurde Heparin-Sepharose® CL-6B als Gelmatrix benutzt. Die Bindungskapazität für das α-Glucosidase-Arg6 Fusionsprotein wurde für beide Gelmaterialien zu 3000 U/ml bestimmt. The cation exchanger Fractogel® EMD SO 3 - -650 was equilibrated with 10 mM potassium phosphate buffer, 1 mM EDTA pH 7.0. Alternatively, Heparin-Sepharose® CL-6B was used as the gel matrix. The binding capacity for the α-glucosidase-Arg6 fusion protein was determined to be 3000 U / ml for both gel materials.
Bestimmung des Beladungsgrades Determination of the degree of loading
Zum Immobilisieren des α-Glucosidase-Arg6 Fusionsproteins an Fractogel® EMD SO3- -650 bzw. Heparin-Sepharose® CL-6B wurde der Rohextrakt (α-Glucosidase Aktivität ca. 200 U/ml) mit einer Pumpgeschwindigkeit von ca. 10 Säulenvolumen/Stunde auf die Säule aufgetragen. Danach wurde die Säule mit 10 mM Kaliumphosphat-Puffer, 1 mM EDTA pH 7,0 gewaschen und die Beladung des Säulenmaterials mit dem α-Glucosidase-Arg6 To immobilize the α-glucosidase-Arg6 fusion protein on Fractogel® EMD SO 3 - -650 or Heparin-Sepharose® CL-6B, the crude extract (α-glucosidase activity approx. 200 U / ml) was pumped at a speed of approx. 10 column volumes / Hour applied to the column. Then the column was 10 mM Potassium phosphate buffer, 1 mM EDTA pH 7.0 and the loading of the column material with the α-glucosidase Arg6
Fusionsprotein bestimmt. Dazu wurde im Säuleneluat (Durchlauf und Waschpuffer) die Gesamtaktivität von nicht an die Säule gebundener α-Glucosidase gemessen und von der auf die Säule aufgetragenen Enzymmenge subtrahiert. Die Bindung des α-Glucosidase-Arg6 Fusionsproteins an Fractogel® SO3- -650 bzw. Fusion protein determined. For this purpose, the total activity of α-glucosidase not bound to the column was measured in the column eluate (flow and washing buffer) and subtracted from the amount of enzyme applied to the column. The binding of the α-glucosidase-Arg6 fusion protein to Fractogel® SO 3 - -650 or
Heparin-Sepharose® CL-6B betrug 95-98 %. Heparin-Sepharose® CL-6B was 95-98%.
B e i s p i e l 4 Example 4
Umsatz von Substrat Sales of substrate
Zur Funktionsprüfung des α-Glucosidase Katalysators wurde die enzymatische Hydrolyseeigenschaft gegenüber den α-Glucosidase-Substraten Maltose und p-NPG bestimmt. Die durch den α- Glucosidase Katalysator pro Zeiteinheit aus Maltose freigesetzte Glucose, bzw. das aus p-NPG freigesetzte Nitrophenol ist ein Maß für die Katalysatorleistung. Die Freisetzung von p-Nitrophenol aus p-NPG wurde im Säuleneluat photometrisch bestimmt (analog α-Glucosidase-Bestimmung). Die Hydrolyse von Maltose wurde im Säuleneluat anhand der freigesetzten Glucose verfolgt. For the functional test of the α-glucosidase catalyst, the enzymatic hydrolysis properties compared to the α-glucosidase substrates maltose and p-NPG were determined. The glucose released from the maltose by the α-glucosidase catalyst per unit of time or the nitrophenol released from p-NPG is a measure of the catalyst performance. The release of p-nitrophenol from p-NPG was determined photometrically in the column eluate (analogous to α-glucosidase determination). The hydrolysis of maltose in the column eluate was followed on the basis of the glucose released.
Glucose-Bestimmung Glucose determination
Testprinzip: Test principle:
Hexokinase  Hexokinase
D-Glucose + ATP ------------------> Glucose-6-P + ADP  D-glucose + ATP ------------------> glucose-6-P + ADP
Glucose-6-Phosphat- Dehydrogenase Glucose-6-phosphate dehydrogenase
Glucose-6-P + NADP+ ------------------> Gluconat-6-P Glucose-6-P + NADP + ------------------> Gluconate-6-P
+ NADPH+H+ Testansatz: + NADPH + H + Test approach:
625 μl Triethanolamin-Puffer (0,3 M TRA, 4 mM MgCl2, pH 7,5) + 50 μl ATP/NADP-Lösung (150 mM ATP, 12 mM NADP in o. g. 625 μl triethanolamine buffer (0.3 M TRA, 4 mM MgCl 2 , pH 7.5) + 50 μl ATP / NADP solution (150 mM ATP, 12 mM NADP in the above
TRA-Puffer)  TRA buffer)
+ 50 μl Glucose enthaltende Probe  + 50 μl sample containing glucose
Ablesen der Extinktion bei 366 nm (E1 ) Reading the absorbance at 366 nm (E 1 )
+ 10 μl Enzym-Gemisch (Glucose 6-Phosphat-Dehydrogenase  + 10 μl enzyme mixture (glucose 6-phosphate dehydrogenase
1 mg/ml und Hexokinase 1 mg/ml, jeweils in 3,2 M Ammoniumsulfat-Lösung).  1 mg / ml and hexokinase 1 mg / ml, each in 3.2 M ammonium sulfate solution).
Nach Stillstand der Reaktion wird E2 bei 366 nm abgelesen. Aus der Differenz E2 - E1 wurde der Glucose-Gehalt der Probe berechnet. After the reaction has stopped, E 2 is read at 366 nm. The glucose content of the sample was calculated from the difference E 2 - E 1 .
Maltose-Hydrolyse mittels nicht-kovalent immobilisierter α- Glucosidase Maltose hydrolysis using non-covalently immobilized α-glucosidase
Maltose-Bioreaktor mit vollständigem Substratumsatz Maltose bioreactor with complete substrate turnover
An einer Heparin-Sepharose® CL-6B-Säule (ca. 3 ml Säulenvolumen) wurden nach dem in Beispiel 3 beschriebenen Verfahren 2500 U α-Glucosidase-Arg6 immobilisiert. Der Substratpuffer enthielt 6 mM Maltose; die Pumpgeschwindigkeit betrug 2500 U α-glucosidase-Arg6 were immobilized on a heparin-Sepharose® CL-6B column (approx. 3 ml column volume) according to the method described in Example 3. The substrate buffer contained 6 mM maltose; the pumping speed was
0,5 ml/min. 0.5 ml / min.
Im kontinuierlichen Betrieb wurde die Hydrolyse von Maltose über einen Zeitraum von 33 Tagen beobachtet (Abbildung 3). Ergebnis: Der Reaktor zeigte während der gesamten Beobachtungszeit 100 %igen Substratumsatz.  In continuous operation, the hydrolysis of maltose was observed over a period of 33 days (Figure 3). Result: The reactor showed 100% substrate conversion during the entire observation period.
Maltose-Bioreaktor mit limitiertem Substratumsatz Maltose bioreactor with limited substrate turnover
An einer Fraktogel® EMD SO3- -650-Säule wurden wie in Beispiel 3 beschrieben 180 U α-Glucosidase-Arg6 immobilisiert. Um den Substratumsatz und die Bindungsstabilität bei verschiedenen Temperaturen zu vergleichen, wurde ein Reaktor bei Raumtemperatur (RT) und ein weiterer identisch beladener Reaktor bei 30 °C betrieben. Der Substratpuffer enthielt 0,15 M Maltose; die Pumpgeschwindigkeit betrug 5 Säulenvolumen/Stunde. 180 U α-glucosidase-Arg6 were immobilized on a Fraktogel® EMD SO 3 - -650 column as described in Example 3. In order to compare the substrate turnover and the binding stability at different temperatures, one reactor was operated at room temperature (RT) and another identically loaded reactor at 30 ° C. The substrate buffer contained 0.15 M maltose; the pumping speed was 5 column volumes / hour.
Der Maltoseumsatz des Bioreaktors wurde über 30 Tage beobachtet. Es zeigte sich während dieses Zeitraumes eine Abnahme von anfangs 50 % auf 38 % (30°C) bzw. von 36 % auf 28 % (RT) (Abbildung 4).  The bioreactor's maltose turnover was observed over 30 days. During this period there was a decrease from initially 50% to 38% (30 ° C) or from 36% to 28% (RT) (Figure 4).
B e i s p i e l 5 Example 5
Renaturierung von denaturiertem α-Glucosidase-Arg6 Fusionsprotein am Träger Renaturation of denatured α-glucosidase-Arg6 fusion protein on the support
Gereinigtes α-Glucosidase-Arg6 Fusionsprotein (ca. 80 U/mg) wurde in 10 mM Kaliumphosphat-Puffer, 1 mM EDTA, 6 M Harnstoff, 2 mM DTE pH 7.0 denaturiert und auf eine mit Denaturierungspuffer äqulibrierte "Tentakelgel"-Säule aufgetragen. Nach Probenauftrag wurde die Säule mit Renaturierungspuffer (10 mM Kaliumphosphat (KPP), 1 mM EDTA, 2 mM 1,4-Dithioerythrit (DTE), pH 7.0) gespült (ca. 10 Säulenvolumen). Die Elution des gebundenen und renaturierten α-Glucosidase-Arg6 Fusionsproteins erfolgte mit 10 mM Kaliumphosphat-Puffer, 1 mM EDTA, 1 M NaCl, pH 7,0. Im mit 1 M NaCl erhaltenen Eluat konnte eine spezifische α-Glucosidase-Aktivität von ca. Purified α-glucosidase-Arg6 fusion protein (approx. 80 U / mg) was denatured in 10 mM potassium phosphate buffer, 1 mM EDTA, 6 M urea, 2 mM DTE pH 7.0 and applied to a "tentacle gel" column equilibrated with denaturing buffer. After sample application, the column was rinsed with renaturation buffer (10 mM potassium phosphate (KPP), 1 mM EDTA, 2 mM 1,4-dithioerythritol (DTE), pH 7.0) (approx. 10 column volumes). The bound and renatured α-glucosidase-Arg6 fusion protein was eluted with 10 mM potassium phosphate buffer, 1 mM EDTA, 1 M NaCl, pH 7.0. In the eluate obtained with 1 M NaCl, a specific α-glucosidase activity of approx.
20 U/mg nachgewiesen werden. 20 U / mg can be detected.
B e i s p i e l 6 Example 6
De- und Renaturierung von nativ gebundener α-Glucosidase-Arg6 am Träger De- and renaturation of natively bound α-glucosidase Arg6 on the support
Das Trägermaterial Fractogel® EMD SO3--650 wurde im Batch- Verfahren mit gereinigtem α-Glucosidase-Arg6 Fusionsprotein (ca. 800 U) beladen, in eine Säule gefüllt, eine Stunde mit Denaturierungspuffer (10 mM KPP, 1 mM EDTA, 2 mM DTE, 6 M Harnstoff, pH 6,8) gespült und anschließend mit Substratpuffer (10 mM KPP, 1 mM EDTA, 0,15 M Maltose, pH 6,8) gewaschen. The carrier material Fractogel® EMD SO 3 --650 was loaded in a batch process with purified α-glucosidase-Arg6 fusion protein (approx. 800 U), filled into a column, for one hour with denaturing buffer (10 mM KPP, 1 mM EDTA, 2 mM DTE, 6M Urea, pH 6.8) and then washed with substrate buffer (10 mM KPP, 1 mM EDTA, 0.15 M maltose, pH 6.8).
Die Renaturierung der α-Glucosidase kann am Anstieg der aus Maltose freigesetzten Glucose im Säuleneluat verfolgt werden (Abbildung 5). The renaturation of α-glucosidase can be followed by the increase in glucose released from maltose in the column eluate (Figure 5).
B e i s p i e l 7 Example 7
Immunologische Bestimmung von Anti HIV Antikörpern unter Verwendung eines Festphasen-gebundenen Antigens Immunological determination of anti HIV antibodies using a solid phase bound antigen
HIV-Antigen HIV antigen
Das HIV Polypeptid HIV2 (envgp32)-HIVl(polp32-envgp41- gagpl7-p24-15-poly(Arg-Lys) ist ein mittels rekombinanter DNA Technologie hergestelltes "multifunktionelles" HIV Fusionspolypeptid. Es besteht aus Protein-Teilbereichen der gag, pol und env Region des HIVl und der env Region des HIV2 Retrovirus. Zudem besitzt das HIV Polypeptid C-terminal eine polykationische Ankersequenz aus 13 positiv geladenen Aminosäuren (Arginin und Lysin). The HIV polypeptide HIV2 (envgp32) -HIVl (polp32-envgp41-gagpl7-p24-15-poly (Arg-Lys) is a "multifunctional" HIV fusion polypeptide produced by means of recombinant DNA technology. It consists of protein segments of the gag, pol and env region of the HIVl and the env region of the HIV2 retrovirus In addition, the HIV polypeptide C-terminal has a polycationic anchor sequence of 13 positively charged amino acids (arginine and lysine).
Die Proteinsequenz des HIV Polypeptides ist in der SEQ ID NO: 2 dargestellt.  The protein sequence of the HIV polypeptide is shown in SEQ ID NO: 2.
Expression des HIV Fusionsproteins in E. coli Expression of the HIV fusion protein in E. coli
Zur Expression des HIV Fusionsproteins wurde der E. coli Stamm RM82+, DSM 5446 (eine Lactose Revertante von RM82) verwendet, der das HIV Fusionsprotein Expressionsplasmid pKK233-2/MYYL_gp32_polp32_gp41_p24-polyArgLys (Ampicillinresistenz) und das dnaY-lacIq Plasmid pUBS500 enthält. Die Konstruktion des HIV-Expressinsplasmids ist in der deutschen Patentanmeldung P 40 02 636.1 vom 30.01.1990 beschrieben. For expression of the HIV fusion protein of the E. coli strain RM82 +, DSM 5446 was used (a lactose revertant of RM82), which contains the HIV fusion protein expression plasmid pKK233-2 / MYYL_gp32_polp32_gp41_p24-polyArgLys (ampicillin resistance) and the dnaY-lacI q plasmid pUBS500. The construction of the HIV expressin plasmid is described in German patent application P 40 02 636.1 dated January 30, 1990.
pUBS500 (EP-A 0 373 365) ist ein pACYC-Derivat (Chang und Cohen, J. Bacteriol. 134 (1978) 1141-1156), das neben der Kanamycinresistenz ein laclq-Repressorgen (Carlos, Nature 274 (1978) 762-765) und ein Gen für die in E. coli seltene t-RNA Arginin (Anticodone: AAG, AGG) besitzt. pUBS500 (EP-A 0 373 365) is a pACYC derivative (Chang and Cohen, J. Bacteriol. 134 (1978) 1141-1156) which, in addition to kanamycin resistance, has a lacl q repressor gene (Carlos, Nature 274 (1978) 762 -765) and a gene for t-RNA arginine (Anticodone: AAG, AGG), which is rare in E. coli.
Zellanzucht Cell culture
Die rekombinanten E. coli Zellen wurden bei 30°C in DYT- Medium (16 g Bactotrypton, 10 g Hefeextrakt, 5 g NaCl pro Liter), supplementiert mit 50 mg/1 Ampicillin plus 50 mg/l Kanamycin, angezogen. Nach Erreichen einer optischen Dichte von 0.6 - 0.8 bei 550 nm wurden die Zellen mit IPTG (Isopropyl-ß-D-thiogalactopyranosid, Endkonzentration 1 mM) induziert. Nach einer Induktionszeit von 4 - 10 Stunden wurden die Zellen abzentrifugiert, mit 10 mM Tris-HCl Puffer, pH 7.0, gewaschen und bis zur Weiterverarbeitung bei -20°C gelagert. The recombinant E. coli cells were grown at 30 ° C. in DYT medium (16 g bactotrypton, 10 g yeast extract, 5 g NaCl per liter), supplemented with 50 mg / 1 ampicillin plus 50 mg / l kanamycin. After reaching an optical density of 0.6-0.8 at 550 nm, the cells were induced with IPTG (isopropyl-β-D-thiogalactopyranoside, final concentration 1 mM). After an induction time of 4 to 10 hours, the cells were centrifuged off, washed with 10 mM Tris-HCl buffer, pH 7.0 and stored at -20 ° C. until further processing.
Zell-Lyse, Solubilisierung des HIV Fusionsproteins Cell lysis, solubilization of the HIV fusion protein
20 g (Naßgewicht) E. coli Zellen wurden in 400 ml 100 mM Tris-HCl Puffer, pH 6.5 - 7.5, resuspendiert, 0.25 mg/ml Lysozym dazugegeben und für 0.5 - 1 Stunde bei Zimmertemperatur inkubiert. Danach wurde die Suspension auf 0 - 4°C gekühlt und die darin enthaltenen Zellen durch Ultraschallbehandlung (French Press) aufgeschlossen. Die Zelltrümmer und das unlösliche aggregierte HIV Fusionsprotein ("inclusion bodies") wurden durch Zentrifugation abgetrennt und das Pellet 1 - 2 mal mit 200 -400 ml 0.5 M NaCl oder KCl und 1 % (v/v) Triton-X-100 gewaschen. Danach wurde das Pellet in 20 ml 50 mM Tris-HCl pH 8.0 mit 8 M Harnstoff und 5 mM ß- Mercaptoethanol unter Rühren (Magnetrührer) bei Zimmertemperatur 1 Stunde resuspendiert und die unlöslichen Zellbestandteile durch Zentrifugation abgetrennt. Die Proteinkonzentration der solubilisierten Proteine wurde nach Bradford (Anal. Biochem. 72 (1976) 248-254) modifiziert nach Gotham et al. (Anal. Biochem. 173 (1988) 353-358) bestimmt. 20 g (wet weight) of E. coli cells were resuspended in 400 ml of 100 mM Tris-HCl buffer, pH 6.5-7.5, 0.25 mg / ml of lysozyme were added and incubated for 0.5-1 hour at room temperature. The suspension was then cooled to 0-4 ° C. and the cells contained therein were disrupted by ultrasonic treatment (French Press). The cell debris and the insoluble, aggregated HIV fusion protein ("inclusion bodies") were separated by centrifugation and the pellet was washed 1-2 times with 200 -400 ml of 0.5 M NaCl or KCl and 1% (v / v) Triton-X-100. After that, the pellet was in Resuspended 20 ml of 50 mM Tris-HCl pH 8.0 with 8 M urea and 5 mM β-mercaptoethanol with stirring (magnetic stirrer) at room temperature for 1 hour and the insoluble cell components were removed by centrifugation. The protein concentration of the solubilized proteins was modified according to Bradford (Anal. Biochem. 72 (1976) 248-254) according to Gotham et al. (Anal. Biochem. 173 (1988) 353-358).
Nicht-kovalente Fixierung des HIV2 (envgp32)-HIV1(polp32- envgp41-gagp17-p24-15-poly(Arg-Lys) Proteins an einer Non-covalent fixation of the HIV2 (envgp32) -HIV1 (polp32-envgp41-gagp17-p24-15-poly (Arg-Lys) protein to one
Gelmatrix  Gel matrix
1 ml in 50 mM Tris-HCl, pH 8.0, und 8 M Harnstoff äquilibrierter Kationenaustauscher Fraktogel® EMD-SO3 -650 (M) wurden mit 100 - 200 mg solubilisiertem HIV Fusionsprotein bei Zimmertemperatur 2 - 6 Stunden unter Schütteln inkubiert. Die nicht an das Gel gebundenen Proteine wurden durch Waschen mit Äquilibrierungspuffer entfernt. Danach wurde das Gel mit 10 mg/ml Rinderserumalbumin in phosphatgepufferter Kochsalzlösung (PBS, 0.15 mM Natriumphosphat; 0.9 % NaCl; pH 7.2) nachinkubiert und mit 0.5 % Tween 20 haltigem demineralisierten Wasser gewaschen. 1 ml of Fraktogel® EMD-SO 3 -650 (M) equilibrated in 50 mM Tris-HCl, pH 8.0, and 8 M urea cation incubator were incubated with 100-200 mg solubilized HIV fusion protein at room temperature for 2 to 6 hours with shaking. The proteins not bound to the gel were removed by washing with equilibration buffer. The gel was then incubated with 10 mg / ml bovine serum albumin in phosphate-buffered saline (PBS, 0.15 mM sodium phosphate; 0.9% NaCl; pH 7.2) and washed with demineralized water containing 0.5% Tween 20.
Bestimmung von Anti-HIV-Antikörpern Determination of anti-HIV antibodies
Die Probe (10 - 100 μl Humanserum) wurde hunderfach in PBS mit 10 % Kälberserumanteil verdünnt und 4 - 12 Stunden bei 37° C mit 10 μl mit HIV Fusionsprotein beschichtetem Gel inkubiert. Anschließend wurde dreimal mit 1 - 1.5 ml Waschlösung (0.5 % Tween 20 in demineralisiertem Wasser) gewaschen. The sample (10-100 ul human serum) was diluted hundreds of times in PBS with 10% calf serum and incubated for 4-12 hours at 37 ° C with 10 ul gel coated with HIV fusion protein. The mixture was then washed three times with 1-1.5 ml of washing solution (0.5% Tween 20 in demineralized water).
Im zweiten Schritt wurde mit einem Konjugat aus Peroxidase und polyklonalem Antikörper (POD-Konjugat, ca. 30 mU Peroxidase/ml in PBS), der gegen den Fcγ-Teil von Human IgG gerichtet ist, für 4 - 12 Stunden inkubiert und dreimal mit Waschlösung gewaschen. Danach wurde 1.5 ml Substratlösung (1.6 mM 2,2'Azino-di-[3- ethylbenzthiazolin-sulfonsäure (6)]-diammoniumsalz (ABTS®); 95 mM Phosphat-Citratpuffer, pH 4.4; 3.1 mM Natriumperborat) zugegeben, 15 - 60 min bei Raumtemperatur inkubiert und die Extinktion in der abzentrifugierten Probe bei 492 nm als Maß für die in der Probe vorhandenen spezifischen Antikörper bestimmt. In the second step, a conjugate of peroxidase and polyclonal antibody (POD conjugate, approx. 30 mU peroxidase / ml in PBS), which is directed against the Fcγ part of human IgG, was incubated for 4 to 12 hours and three times with washing solution washed. Then 1.5 ml of substrate solution (1.6 mM 2,2'-azino-di- [3-ethylbenzthiazoline-sulfonic acid (6)] - diammonium salt (ABTS®); 95 mM phosphate citrate buffer, pH 4.4; 3.1 mM sodium perborate) was added, 15 - Incubated for 60 min at room temperature and the absorbance in the centrifuged sample at 492 nm was determined as a measure of the specific antibodies present in the sample.
Normalseren zeigten im Vergleich mit Anti-HIV Poolseren eine relative Signalstärke (OD492 Anti-HIV-Poolserum/OD492 Normalserum) von 7 - 50. (OD492 : Extinktion bei 492 nm) In comparison with anti-HIV pool sera, normal sera showed a relative signal strength (OD 492 anti-HIV pool serum / OD 492 normal serum) of 7-50. (OD 492 : extinction at 492 nm)
Nachstehend sind die Hinterlegungsangaben für die erwähnten Zellinien angeführt. The deposit information for the cell lines mentioned is given below.
DSM ♤ DEUTSCHE SAMMLUNG VON MIKROORGANISMENDSM ♤ GERMAN COLLECTION OF MICROORGANISMS
German Collection of Microorganisms German Collection of Microorganisms
GESELLSCHAFT FÜR BlOTECHNOLOGISCHE FORSCHUNG MBH  SOCIETY FOR BLOTECHNOLOGICAL RESEARCH MBH
DSM · Gnsebachstrasse 8 · D-3400 Göttingen, Germany Tel. (0551) 393822/393823 DSMGnsebachstrasse 8D-3400 Göttingen, Germany Tel. (0551) 393822/393823
Boehringer Mannheim GmbH Datum/Date 07.05.1986 Biochemica Werk Tutzing Boehringer Mannheim GmbH Datum / Date 07.05.1986 Biochemica plant Tutzing
Postfach 1263/64 Box 1263/64
8132 TUTZING  8132 TUTZING
EMPFANGSBESTÄTIGUNG ACKNOWLEDGMENT OF RECEIPT
Wir bestätigen, daß das Plasmid mit dem vom Hinterleger zugeteilten Bezugszeichen We confirm that the plasmid has the reference number assigned by the depositor
pePa 119  pePa 119
unter der Eingangsnummer DSM 3691P am 09.04.1986 in der DSM hinterlegt wurde.
Figure imgf000025_0001
was deposited in the DSM on April 9, 1986 under the entry number DSM 3691P.
Figure imgf000025_0001
DEUTSCHE SAMMLUNG VON MIKROORGANISMEN GERMAN COLLECTION OF MICROORGANISMS
DSM ♤ DEUTSCHE SAMMLUNG VON MIKROORGANISMEN DSM ♤ GERMAN COLLECTION OF MICROORGANISMS
German Collection of Microorganlsms  German Collection of Microorganlsms
GESELLSCHAFT FÜR BIOTECHNOLOGISCHE FORSCHUNG MBH  SOCIETY FOR BIOTECHNOLOGICAL RESEARCH MBH
DSM · Grisebachstrasse 8 · D-3400 Göttingen, Germany Tel.(0551)393827 (Sekretariat)/3938-DSMGrisebachstrasse 8D-3400 Göttingen, Germany Tel. (0551) 393827 (Secretariat) / 3938-
Boehringer Mannheim GmbH Boehringer Mannheim GmbH
Biochemica Werk Tutzing  Biochemica plant Tutzing
Abt. E - B 1  Dept. E - B 1
Postfach 1263/64  Box 1263/64
8132 TUTZING  8132 TUTZING
IhrZeichen /Your ref. Unser Zeichen / Our ref. Datum / Date Your sign / Your ref. Our sign / Our ref. Date
38687 14.07.1987  38687 Jul 14, 1987
EMPFANGSBESTÄTIGUNG ACKNOWLEDGMENT OF RECEIPT
Wir bestätigen, daß das Plasmid mit dem unten genannten vom Hinterleger zugeteilten Bezugszeichen We confirm that the plasmid has the reference number given below by the depositor
YRp-Gluc.pl unter der Eingangsnummer YRp-Gluc.pl under the entry number
DSM 4173 P am 29.06.1987 in der DSM hinterlegt wurde. DSM 4173 P was deposited in the DSM on June 29, 1987.
Figure imgf000026_0001
Figure imgf000026_0001
DEUTSCHE SAMMLUNG VON MIKROORGANISMEN DSM ♤ DEUTSCHE SAMMLUNG VON MIKROORGANISMEN GERMAN COLLECTION OF MICROORGANISMS DSM ♤ GERMAN COLLECTION OF MICROORGANISMS
German Collection of Mlcroorganisms  German Collection of Mlcroorganisms
GESELLSCHAFT FÜR BIOTECHNOLOGISCHE FORSCHUNG MBH  SOCIETY FOR BIOTECHNOLOGICAL RESEARCH MBH
DSM · Grisebachstrasse 8 · D-3400 Göttingen, Germany Tel. (0551) 393827 (Sekretariat)/3938- DSMGrisebachstrasse 8D-3400 Göttingen, Germany Tel. (0551) 393827 (Secretariat) / 3938-
BOEHRINGER MANNHEIM GMBH BOEHRINGER MANNHEIM GMBH
Biochemica Werk Tutzing  Biochemica plant Tutzing
Abt. E - B 1  Dept. E - B 1
Postfach 1263/64  Box 1263/64
8132 Tutzing 8132 Tutzing
Figure imgf000027_0001
Figure imgf000027_0001
Ihr Zeichen / Your ref. Unser Zeichen / Our ref. Datum / Date Your sign / Your ref. Our sign / Our ref. Date
Bk-B1 39087 14.07.1987  Bk-B1 39087 07/14/1987
Verwendung eines DSM-Stammes in einer Patentanmeldung  Use of a DSM strain in a patent application
Bestätigung  confirmation
Wir bestätigen, daß der unten aufgeführte Mikroorganismus in der allgemeinen Sammlung der DSM vorhanden ist. Die Aufbewahrung des Stammes erfolgt für mindestens 30 weitere Jahre ab Datum dieser Bestätigung. Die Frist berechnet sich ab dem Tag der Offenlegung, falls auf den Stamm bei Anmeldung des Patents Bezug genommen und dies der DSM zur Kenntnis gegeben wurde.  We confirm that the microorganism listed below is present in the general collection of the DSM. The trunk is kept for at least 30 more years from the date of this confirmation. The deadline is calculated from the date of disclosure if the strain was referred to when the patent was registered and the DSM was informed of this.
Darüber hinaus wird die Kultur mindestens bis zum Ablauf von 5 Jahren nach Eingang des letzten Antrags auf Abgabe einer Probe aufbewahrt.  In addition, the culture is kept for at least 5 years after receipt of the last application for a sample.
Vermehrungsfähige Proben dieser Kultur wurden und werden während des gesamten weiteren Zeitraums nach Maßgabe der nationalen Bestimmungen über den Verkehr mit Mikroorganismen an jedermann abgegeben.  Reproductive samples of this culture have been and will be given to everyone throughout the rest of the period in accordance with national regulations governing the circulation of microorganisms.
Eine Prüfung der Lebensfähigkeit wurde am unten genannten Datum durchgeführt. Zu diesem Zeitpunkt war der Mikroorganismus lebensfähig.  A viability test was performed on the date below. At that point the microorganism was viable.
Taxonomische BezeichDSM- Tag der Aufnahme Datum Tag der Offennung des Mikroorganismus Nummer in die DSM Lsbensfä- legung nach An- higkeits- gabe des Anmel- prüfung ders  Taxonomic designation DSM day of admission Date day of the microorganism's number number in the DSM lifespan according to the details of the registration test
Escherichia coli HB101 1607 16.07.1979 09.03.1987  Escherichia coli HB101 1607 July 16, 1979 March 9, 1987
Micromonospora edhinospora 43141 15.04.1975 10.07.1987 Micromonospora edhinospora 43141 April 15, 1975 July 10, 1987
Figure imgf000027_0002
Figure imgf000027_0002
Deutsche Sammlung  German collection
von Mikroorganismen
Figure imgf000028_0001
Figure imgf000029_0001
SEQ ID NO:1 of microorganisms
Figure imgf000028_0001
Figure imgf000029_0001
SEQ ID NO: 1
ART DER SEQUENZ: Nukleotidsequenz  TYPE OF SEQUENCE: nucleotide sequence
SEQUENZLÄNGE : 4648  SEQUENCE LENGTH: 4648
STRANGFORM: Einzelstrang  STRAND FORM: Single strand
Plasmid Pkk177-3/GLUCPI_ARG6 Plasmid Pkk177-3 / GLUCPI_ARG6
1 GGATCCAGCT TATCGACTGC ACGGTGCACC AATGCTTCTG GCGTCAGGCA GCCATCGGAA 1 GGATCCAGCT TATCGACTGC ACGGTGCACC AATGCTTCTG GCGTCAGGCA GCCATCGGAA
61 GCTGTGGTAT GGCTGTGCAG GTCGTAATC ACTGCATAAT TCGTGTCGCT CAAGGCGCAC 61 GCTGTGGTAT GGCTGTGCAG GTCGTAATC ACTGCATAAT TCGTGTCGCT CAAGGCGCAC
121 TCCCGTTCTG GATATGTTT TTTGCGCCGA CATCATAACG GTTCTGGCAA ATATTCTGAA 121 TCCCGTTCTG GATATGTTT TTTGCGCCGA CATCATAACG GTTCTGGCAA ATATTCTGAA
181 ATGAGCTGTT GACAATTAAT CATCGGCTCG TATAATGTGT GGAATTGTGA GCGGATAACA 181 ATGAGCTGTT GACAATTAAT CATCGGCTCG TATAATGTGT GGAATTGTGA GCGGATAACA
241 ATTTCACACA GGAAACAGAA TTATGACGKT ATCCGATCAT CCAGAAACAG AACCAABGTG 301 GTGGAAAGAG GCCACAATCT ATCAAATTTA CCCAGCAAGT TTTAAAGACT CCAATAACGA 241 ATTTCACACA GGAAACAGAA TTATGACGKT ATCCGATCAT CCAGAAACAG AACCAABGTG 301 GTGGAAAGAG GCCACAATCT ATCAAATTTA CCCAGCAAGT TTTAAAGACT CCAATAACGA
361 TGGCTGGGGT GATTTAAAAG GTATCACTTC CAAGTTGCAG TA TATTAAAG ATCTTGGCGT 421 TCATGCTATT TGGGTTGTC CGTTTTATGA CTCTCCTCAA CAAGATATGG GGTATGATAT 361 TGGCTGGGGT GATTTAAAAG GTATCACTTC CAAGTTGCAG TA TATTAAAG ATCTTGGCGT 421 TCATGCTATT TGGGTTGTC CGTTTTATGA CTCTCCTCAA CAAGATATGG GGTATGATAT
481 ATCTAACTAC GAAAAGGTCT GGCCCACATA CGG TACCAAC GAGGACTGTT TTGAGCTAAT 481 ATCTAACTAC GAAAAGGTCT GGCCCACATA CGG TACCAAC GAGGACTGTT TTGAGCTAAT
541 TGACAAGACT CATAAGCTGG GTATGAAATT CACACCGAT TTGGTTATCA ACCACTGTTC 541 TGACAAGACT CATAAGCTGG GTATGAAATT CACACCGAT TTGGTTATCA ACCACTGTTC
601 TACAGAACAC GAATGGTTCA AAGAGAGCAG ATCCTCGAAG ACCAATCCGA AGCGTGACTG 661 GTTCTTCTGGAGACCTCCTAAGGGTTATGACGCCGAAGGC AAGCCAATTC CTCCAAACAA 601 TACAGAACAC GAATGGTTCA AAGAGAGCAG ATCCTCGAAG ACCAATCCGA AGCGTGACTG 661 GTTCTTCTGGAGACCTCCTAAGGGTTATGACGCCGAAGGC AAGCCAATTC CTCCAAACAA
721 TTGGAAATCT TTCTTTGGTGGTTCAGCTTGGACTTTTGAT GAAACTACAA ATGAATTTTA 721 TTGGAAATCT TTCTTTGGTGGTTCAGCTTGGACTTTTGAT GAAACTACAA ATGAATTTTA
781 CCTCCGTTTGTTTGCGAGTC GTCAAGTTGA CTTGAATTGGGAGAATGAAG ACTGCAGAAG 841 GGCAATCTTT GAAAGTCCTGTTGGATTTTGGCTGGACCAT GGTGTAGATG GTTTTAGAAT 781 CCTCCGTTTGTTTGCGAGTC GTCAAGTTGA CTTGAATTGGGAGAATGAAG ACTGCAGAAG 841 GGCAATCTTT GAAAGTCCTGTTGGATTTTGGCTGGACCAT GGTGTAGATG GTTTTAGAAT
901 CGATACCGCT GGTTTGTATT CGAAACGTCC TGGTTTACCA GATTCCCCAA TTTTTGACAA 961 AACCTCGAAA TTACAACATC CAAATTGGGG GTCTCACAAT GGT CCTAGGA TTCATGAATA901 CGATACCGCT GGTTTGTATT CGAAACGTCC TGGTTTACCA GATTCCCCAA TTTTTGACAA 961 AACCTCGAAA TTACAACATC CAAATTGGGG GTCTCACAAT GGT CCTAGGA TTCATGAATA
1021 TCATCAAGAA CTACACAGAT TTATGAAAAA CAGGGTGAAA GATGGTAGAG AAATAATGAC1021 TCATCAAGAA CTACACAGAT TTATGAAAAA CAGGGTGAAA GATGGTAGAG AAATAATGAC
1081 AGTCGGTGAA GTTGCCCATG GAAGTGATAA TGCTTTATAC ACCAGTGCAG CTAGATACGΑ1081 AGTCGGTGAA GTTGCCCATG GAAGTGATAA TGCTTTATAC ACCAGTGCAG CTAGATACGΑ
1141 AGTCAGCGAA GTTTTCTCCT TCACGCACGT TGAAGTTGGT ACCTCGCCAT TTTTCCGTTA1141 AGTCAGCGAA GTTTTCTCCT TCACGCACGT TGAAGTTGGT ACCTCGCCAT TTTTCCGTTA
1201 TAACATAGTG CCCTTCACCT TGAAACAATG GAAAGAAGCC ATTGCATCGA ACTTTTTGTT1201 TAACATAGTG CCCTTCACCT TGAAACAATG GAAAGAAGCC ATTGCATCGA ACTTTTTGTT
1261 CATTAACGGT ACTGATAGTT GGGCTACCAC CTACATCGAG AATCACGATC AAGCCCGGTC1261 CATTAACGGT ACTGATAGTT GGGCTACCAC CTACATCGAG AATCACGATC AAGCCCGGTC
1321 AATTACGAGA TTTGCTGACG ATTCGCCAAA GTACCGTAAA ATATCTGGTA AGCTGTTAAC1321 AATTACGAGA TTTGCTGACG ATTCGCCAAA GTACCGTAAA ATATCTGGTA AGCTGTTAAC
1381 ATTGCTAGAA TGTTCATTGA CAGGTACGTT GTATGTCTAT CAAGGTCAGG AGATAGGCCA1381 ATTGCTAGAA TGTTCATTGA CAGGTACGTT GTATGTCTAT CAAGGTCAGG AGATAGGCCA
1441 GATCAATTTC AAGGAATGGC CTATTGAAAA GTATGAGGAC GTTGATGTGΑ AAAACAACTA1441 GATCAATTTC AAGGAATGGC CTATTGAAAA GTATGAGGAC GTTGATGTGΑ AAAACAACTA
1501 CGAGATTATC AAAAAAAGTT TTGGTAΑAAA CTCGAAGGAA ATGAAGGATT TTTTAAAGG1501 CGAGATTATC AAAAAAAGTT TTGGTAΑAAA CTCGAAGGAA ATGAAGGATT TTTTAAAGG
1561 AATCGCCCTA CTTTCTAGAG ATCATTCGAG AACTCCCATG CCATGGACGA AAGATAAGCC1561 AATCGCCCTA CTTTCTAGAG ATCATTCGAG AACTCCCATG CCATGGACGA AAGATAAGCC
1621 CAATGCTGGA TTTACTGGCC CAGATGTTAA ACCTTGGTTT TTCTTGAATG AATCTTTGA1621 CAATGCTGGA TTTACTGGCC CAGATGTTAA ACCTTGGTTT TTCTTGAATG AATCTTTGA
1681 GCAAGGAATC AATGTTGAGC AGGAATCCAG AGATGATGAC TCAGTTCTCA ATTTTTGGAA1681 GCAAGGAATC AATGTTGAGC AGGAATCCAG AGATGATGAC TCAGTTCTCA ATTTTTGGAA
1741 AAGGGCCTTG CAAGCCAGAA AGAAATATAA GGAACTTATG ATTTATGGTT ACGATTTCCA1741 AAGGGCCTTG CAAGCCAGAA AGAAATATAA GGAACTTATG ATTTATGGTT ACGATTTCCA
1801 ATTCATTGAT TTAGACAGTG ACCAGATCTT TAGCTTCACT AAAGAGTACG GAGACAAGAC1801 ATTCATTGAT TTAGACAGTG ACCAGATCTT TAGCTTCACT AAAGAGTACG GAGACAAGAC
1861 GCTGTTTGCT GCTTTGAATT TCAGTGGCGA AGAAATTGAA TTCAGCCTCC CAAGAGAAGG1861 GCTGTTTGCT GCTTTGAATT TCAGTGGCGA AGAAATTGAA TTCAGCCTCC CAAGAGAAGG
1921 TGLTTCTTTA TCTTTTATTC TTGGAAATTA TGATGATACT GACGTTTCCT CCAGAGTTTT 1981 GAAACCATGG GAAGGTAGAA TCTACCTGGT CAAAGGGCGC CGACGTCGCC GGCGTTAATA 2041 AGCTTCTGTT TTGGCGGATG AGAGAAGATT TTCAGCCTGA TACAGATTAA ATCAGAACGC 2101 AGAAGCGGTC TGATAAAACA GAATTTGCCT GGCGGCAGTA GCGCGGTGGT CCCACCTGAC1921 TGLTTCTTTA TCTTTTATTC TTGGAAATTA TGATGATACT GACGTTTCCT CCAGAGTTTT 1981 GAAACCATGG GAAGGTAGAA TCTACCTGGT CAAAGGGCGC CGACGTCGCC GGCGTTAATA 2041 AGCTTCTGTATTGGAGACAGATTTGAGAGTAAGAT 2101 AGAAGCGGTC TGATAAAACA GAATTTGCCT GGCGGCAGTA GCGCGGTGGT CCCACCTGAC
2161 CCCATGCCGA ACTCAGAAGT GAAACGCCGT AGCGOCGATG GEAGTGTGGG CTCTCCCCAT2161 CCCATGCCGA ACTCAGAAGT GAAACGCCGT AGCGOCGATG GEAGTGTGGG CTCTCCCCAT
2221 GCGAGAGTAG GGAACTGCCA GGCATCAAAT AAAACGAAAG GCTCAGTGGA AAGACTGGGC2221 GCGAGAGTAG GGAACTGCCA GGCATCAAAT AAAACGAAAG GCTCAGTGGA AAGACTGGGC
2281 CTTTCGTTTT ATCTGTTGTT TGTCGGTGAA CGCTCTCCTG AGEAGGACAA ATCCGCCGGG2281 CTTTCGTTTT ATCTGTTGTT TGTCGGTGAA CGCTCTCCTG AGEAGGACAA ATCCGCCGGG
2341 AGCGGATTTG AACGTTGCGA AGCAACGGCC CGGAGGGTGG CGGGCAGGAC GCCCGCCATA2341 AGCGGATTTG AACGTTGCGA AGCAACGGCC CGGAGGGTGG CGGGCAGGAC GCCCGCCATA
2401 AACTGCCAGG CATCAAATTA AGCAGAAGGC CATGCTGACG GATGGCCTTT TTGCGTTTCT2401 AACTGCCAGG CATCAAATTA AGCAGAAGGC CATGCTGACG GATGGCCTTT TTGCGTTTCT
2461 ACAAACTCTT TTGTTTATTT TTCTAAATAC ATTCAAATAT GTATCCGCTC ATGAGACAAT2461 ACAAACTCTT TTGTTTATTT TTCTAAATAC ATTCAAATAT GTATCCGCTC ATGAGACAAT
2521 AACCCTGATA AATGCTTCAA TAATATTGAA AAAGGAAGAG TATGAGEATT CAACATTTCC2521 AACCCTGATA AATGCTTCAA TAATATTGAA AAAGGAAGAG TATGAGEATT CAACATTTCC
2581 GTGTCGCCCT TATTCCCTTT TTTGCGGCAT TTTGCCTTCC TGTTTTTGCT CACCCAGAAA2581 GTGTCGCCCT TATTCCCTTT TTTGCGGCAT TTTGCCTTCC TGTTTTTGCT CACCCAGAAA
2641 CGCTGCTGAA AGTAAAAGAT GCTGAAGATC AGTTGGGTGC ACGACTGGGT TACATCGAAC2641 CGCTGCTGAA AGTAAAAGAT GCTGAAGATC AGTTGGGTGC ACGACTGGGT TACATCGAAC
2701 TGGATCTCAA CAGCGGTAAG ATCCTTGAGA CTTTTCGCCC CGAAGAACGT TTTCCAATGA2701 TGGATCTCAA CAGCGGTAAG ATCCTTGAGA CTTTTCGCCC CGAAGAACGT TTTCCAATGA
2761 TGAGCACTTT TAAAGTTCTG CTATGTGGCG CGGTATTATC CCGTGTTGAC GCCGGGCAAG2761 TGAGCACTTT TAAAGTTCTG CTATGTGGCG CGGTATTATC CCGTGTTGAC GCCGGGCAAG
2821 AGCAACTCGG TCGCCGCATA CACTATTCTC AGAATGACTT GGTTGAGTAC TCACCAGTCA2821 AGCAACTCGG TCGCCGCATA CACTATTCTC AGAATGACTT GGTTGAGTAC TCACCAGTCA
2881 CAGAAAAGCA TCTTACGGAT GGCATGACAG TAAGAGAATT ATGCAGTGCT GCCATAACCA2881 CAGAAAAGCA TCTTACGGAT GGCATGACAG TAAGAGAATT ATGCAGTGCT GCCATAACCA
2941 TGAGTGATAA CACTGCGGCC AACTTACTTC TGACAACGAT CGGAGGACCG AAGGAGCTAA2941 TGAGTGATAA CACTGCGGCC AACTTACTTC TGACAACGAT CGGAGGACCG AAGGAGCTAA
3001 CCGCTTTTTT GCACAACATG GGGGATATG TAACTCGCCT TGATCGTTGG GAACCGGAGC3001 CCGCTTTTTT GCACAACATG GGGGATATG TAACTCGCCT TGATCGTTGG GAACCGGAGC
3061 TGAATGAAGC CA TACCAAAC GACGAGCGTG ACACCACGAT GCCTGCAGCA ATGGCAACAA3061 TGAATGAAGC CA TACCAAAC GACGAGCGTG ACACCACGAT GCCTGCAGCA ATGGCAACAA
3121 CGTTGCGCAA ACTAITAACT GGCGAACTAC TTACTCTAGC TTCCCGGCAA CAATTAATAG3121 CGTTGCGCAA ACTAITAACT GGCGAACTAC TTACTCTAGC TTCCCGGCAA CAATTAATAG
3181 ACTGGATGGA GGCGGATAAA GTTGCAGGAC CACTTCTGCG CTCGGCCCTT CCGGCTGGCT 3241 GGTTTATTGC TGATAAATCT GGAGCCGGTG AGCGTGGGTC TCGCGGTATC ATTGCAGCAC3181 ACTGGATGGA GGCGGATAAA GTTGCAGGAC CACTTCTGCG CTCGGCCCTT CCGGCTGGCT 3241 GGTTTATTGC TGATAAATCT GGAGCCGGTG AGCGTGGGTC TCGCGGTATC ATTGCAGCAC
3301 TGGGGCCAGA TGGTAAGCCC TCCCGTATCG TAGTTATCTA CACGACGGGG AGTCAGGCAA3301 TGGGGCCAGA TGGTAAGCCC TCCCGTATCG TAGTTATCTA CACGACGGGG AGTCAGGCAA
3361 CTATGGATGA ACGAAATAGA CAGATCGCTG AGATAGGTGC CTCACTCATT AAGCATTGGT3361 CTATGGATGA ACGAAATAGA CAGATCGCTG AGATAGGTGC CTCACTCATT AAGCATTGGT
3421 AACTGTCAGA CCAAGTTTAC TCATATATAC TTTAGATTGA TTTAAAACTT CATTTTTAAT3421 AACTGTCAGA CCAAGTTTAC TCATATATAC TTTAGATTGA TTTAAAACTT CATTTTTAAT
3481 TTAAAAGGAT CTAGGTGAAG ATCCTTTTTG ATAATCTCAT GACCAAAATC CCTTAACGTG3481 TTAAAAGGAT CTAGGTGAAG ATCCTTTTTG ATAATCTCAT GACCAAAATC CCTTAACGTG
3541 AGTTTTCGTT CCACTGAGCG TCAGACCCCG TAGAAAAGAT CAAAGGATCT TCTTGAGATC3541 AGTTTTCGTT CCACTGAGCG TCAGACCCCG TAGAAAAGAT CAAAGGATCT TCTTGAGATC
3601 CTTTTTTTCT GCGCGEAATC TGCTGCTTGC AAACAAAAAA ACCACCGCTA CCAGCGGTGG3601 CTTTTTTTCT GCGCGEAATC TGCTGCTTGC AAACAAAAAA ACCACCGCTA CCAGCGGTGG
3661 TTTGTTTGCC GGATCAAGAG CTACCAACTC TTTTTCCGAA GGTAACTGGC TTCAGCAGAG3661 TTTGTTTGCC GGATCAAGAG CTACCAACTC TTTTTCCGAA GGTAACTGGC TTCAGCAGAG
3721 CGCAGATACC AAATACTGTC CTTCTAGTGT AGCCCTAGTT AGGCCACCAC TTCAAGAACT3721 CGCAGATACC AAATACTGTC CTTCTAGTGT AGCCCTAGTT AGGCCACCAC TTCAAGAACT
3781 CTGTAGCACC GCCEACATAC CTCGCTCTGC TAATCCTGTT ACCAGTGGCT GCTGCCAGTG3781 CTGTAGCACC GCCEACATAC CTCGCTCTGC TAATCCTGTT ACCAGTGGCT GCTGCCAGTG
3841 GCGATAAGTC GTGTCTTACC GGGTTGGACT CAAGACGATA GTTACCGGAT AAGGCGCAGC3841 GCGATAAGTC GTGTCTTACC GGGTTGGACT CAAGACGATA GTTACCGGAT AAGGCGCAGC
3901 GGTCGGQCTG AACGGQGGGT TCGTGCACAC AGCCCAGCTT GGAGCGAACG ACCTACACCG3901 GGTCGGQCTG AACGGQGGGT TCGTGCACAC AGCCCAGCTT GGAGCGAACG ACCTACACCG
3961 AACTGAGATA CCTACAGCGT GAGCATTGAG AAAGCGCCAC GCTTCCCGAA GGGAGAAAGG3961 AACTGAGATA CCTACAGCGT GAGCATTGAG AAAGCGCCAC GCTTCCCGAA GGGAGAAAGG
4021 CGGACAGGTA TCCGGTAAGC GGCAGGGTCG GAACAGGAGA GCGCACGAGG GAGCTTCCAG4021 CGGACAGGTA TCCGGTAAGC GGCAGGGTCG GAACAGGAGA GCGCACGAGG GAGCTTCCAG
4081 GGGGAAACGC CTGGTATCTT TATAGTCCTG TCGGGTTTCG CC-ACCTCTGA CTTGAGCGTC4081 GGGGAAACGC CTGGTATCTT TATAGTCCTG TCGGGTTTCG CC-ACCTCTGA CTTGAGCGTC
4141 GATTTTTGTG ATGCTCGTCA GGGGGGCGGA GCCTATGGAA AAACGCCAGC AACGCGGCCT4141 GATTTTTGTG ATGCTCGTCA GGGGGGCGGA GCCTATGGAA AAACGCCAGC AACGCGGCCT
4201 TTTTACGGTT CCTGGCCTTT TGCTGGCCTT TTGCTCACAT GTTCTTTCCT GCGTTATCCC4201 TTTTACGGTT CCTGGCCTTT TGCTGGCCTT TTGCTCACAT GTTCTTTCCT GCGTTATCCC
4261 CTGATTCTGT GGATAACCGT ATTACCGCCT TT GAGTGAGC TGATACCGCT CGCCGCAGCC4261 CTGATTCTGT GGATAACCGT ATTACCGCCT TT GAGTGAGC TGATACCGCT CGCCGCAGCC
4321 GAACGACCGA GCGCAGCGAG TCAGTGAGCG AGGAAGCGGA AGAGCGCCTG ATGCGGTATT 4381 TTCTCCTTAC GCATCTGTGC GGTATTTCAC ACCGCATATG GTGCACTCTC AGTACAATCT4321 GAACGACCGA GCGCAGCGAG TCAGTGAGCG AGGAAGCGGA AGAGCGCCTG ATGCGGTATT 4381 TTCTCCTTAC GCATCTGTGC GGTATTTCAC ACCGCATATG GTGCACTCTC AGTACAATCT
4441 GCTCTGATGC CGCATAGTTA AGCCAGTATA CACTCCGCTA TCGCTACGTG ACTGGGTCAT4441 GCTCTGATGC CGCATAGTTA AGCCAGTATA CACTCCGCTA TCGCTACGTG ACTGGGTCAT
4501 GGCTGCGCCC CGACACCCGC CAACACCCGC TCACGCGCCC TGACGGGCTT GTCTGCTCCC4501 GGCTGCGCCC CGACACCCGC CAACACCCGC TCACGCGCCC TGACGGGCTT GTCTGCTCCC
4561 GGCATCCGCT TACAGACAAG CTSTGACCGT CTCCGGGAGC TGCATGTGTC AGAGGTTTTC4561 GGCATCCGCT TACAGACAAG CTSTGACCGT CTCCGGGAGC TGCATGTGTC AGAGGTTTTC
4621 ACCGTCATCA CGGAAACGCG CGAGGCAG 4621 ACCGTCATCA CGGAAACGCG CGAGGCAG
SEQ ID NO: 2 SEQ ID NO: 2
ART DER SEQUENZ:Aminosäuresequenz  TYPE OF SEQUENCE: amino acid sequence
SEQUENZLÄNGE : 770 Aminosäuren  SEQUENCE LENGTH: 770 amino acids
STRANGFORM:Einzelstrang  STRAND FORM: Single strand
Met Tyr Tyr Leu Glu Phe Gln Gln Gln Gln Gln Leu Leu Asp Val Val 16 Lys Arg Gln Gln Glu Leu Leu Arg Leu Thr Val Trp Gly Thr Lys Asn 32 Leu Gln Ala Arg Val Thr Ala Ile Glu Lys Tyr Leu Gln Asp Gln Ala 48 Arg Leu Asn Ser Trp Gly Cys Ala Phe Arg Gln Val Cys His Thr Thr 64 Val Pro Trp Val Asn Asp Ser Leu Ala Pro Asp Trp Asp Asn Met Thr 80 Trp Gln Glu Trp Glu Lys Gln Val Arg Tyr Leu Glu Ala Asn Ile Ser 96 Lys Ser Leu Glu Gln Ala Gln Ile Gln Gln Glu Lys Asn Met Tyr Glu 112 Leu Gln Lys Leu Asn Ser Trp Asp Asp Pro Leu Glu Ser Cys Asp Lys 128 Cys Gln Leu Lys Gly Glu Ala Met His Gly Gln Val Asp Cys Ser Pro 144 Gly Ile Trp Gln Leu Asp Cys Thr His Leu Glu Gly Lys Ile Ile Leu 160 Val Ala Val His Val Ala Ser Gly Tyr Ile Glu Ala Glu Val Ile Pro 176 Ala Glu Thr Gly Gln Glu Thr Ala Tyr Phe Ile Leu Lys Leu Ala Gly 192 Arg Trp Pro Val Lys Val Ile His Thr Asp Asn Gly Ser Asn Phe Thr 208 Ser Thr Thr Val Lys Ala Ala Cys Trp Trp Ala Gly Ile Lys Gln Glu 224 Phe Gly Ile Pro Tyr Asn Pro Gln Ser Gln Gly Val Val Glu Ser Met 240 Asn Lys Glu Leu Lys Lys Ile Ile Gly Gln Val Arg Asp Gln Ala Glu 256 His Leu Lys Thr Ala Val Gln Met Ala Val Phe Ile His Asn Phe Lys 272 Arg Lys Gly Gly Ile Gly Gly Tyr Ser Ala Gly Glu Arg Ile Val Asp 288 Ile Ile Ala Thr Asp Ile Gln Thr Lys Glu Leu Gln Lys Gln Ile Ile 304 Lys Ile Gln Asn Phe Arg Val Tyr Tyr Arg Asp Ser Arg Asp Pro Leu 320 Trp Lys Gly Pro Ala Lys Leu Leu Trp Lys Gly Glu Gly Ala Val Val 336 Ile Gln Asp Asn Ser Glu Ile Lys Val Val Pro Arg Arg Lys Ala Lys 352 Ile Ile Arg Asp Tyr Gly Lys Gln Ser Asp Arg Ser Ser Arg Val Gln 368 Thr Arg Gln Leu Leu Ser Gly Ile Val Gln Gln Gln Asn Asn Leu Leu 384 Arg Ala Ile Glu Thr Gln Gln His Leu Leu Gln Leu Thr Val Trp Gly 400 Ile Lys Gln Leu Gln Ala Arg Val Leu Ala Val Glu Arg Tyr Leu Gln 416 Asp Gln Arg Leu Leu Gly Ile Trp Gly Cys Ser Gly Lys Leu Ile Cys 432 Thr Thr Thr Val Pro Trp Asn Thr Ser Trp Ser Asn Lys Ser Leu Asp 448 Thr Ile Trp His Asn Met Thr Trp Met Glu Trp Glu Arg Glu Ile Asp 464 Asn Tyr Thr Ser Ser Asp Lys Gly Asn Ser Ser Gln Val Ser Gln Asn 480 Tyr Pro Ile Val Gln Asn Leu Gln Gly Gln Met Val His Gln Ala Ile 496 Ser Pro Arg Thr Leu Asn Ala Trp Val Lys Val Ile Glu Glu Lys Ala 512 Phe Ser Pro Glu Val Ile Pro Met Phe Ser Ala Leu Ser Glu Gly Ala 528 Thr Pro Gln Asp Leu Asn Thr Met Leu Asn Thr Val Gly Gly His Gln 544 Ala Ala Met Gln Met Leu Lys Glu Thr Ile Asn Glu Glu Ala Ala Glu 560 Trp Asp Arg Val His Pro Val His Ala Gly Pro Ile Ala Pro Gly Gln 576 Met Arg Glu Pro Arg Gly Ser Asp Ile Ala Gly Thr Thr Ser Thr Leu 592 Gln Glu Gln Ile Gly Trp Met Thr Asn Asn Pro Pro Ile Pro Val Gly 608 Glu Ile Tyr Lys Arg Trp Ile Ile Leu Gly Leu Asn Lys Ile Val Arg 624 Met Tyr Ser Pro Val Ser Ile Leu Asp Ile Arg Gln Gly Pro Lys Glu 640 Pro Phe Arg Asp Tyr Val Asp Arg Phe Tyr Lys Thr Leu Arg Ala Glu 656 Gln Ala Ser Gln Glu Val Lys Asn Trp Met Thr Glu Thr Leu Leu Val 672 Gln Asn Ala Asn Pro Asp Cys Lys Thr Ile Leu Lys Ala Leu Gly Pro 688 Ala Ala Thr Leu Glu Glu Met Met Thr Ala Cys Gln Gly Val Gly Gly 704 Pro Gly His Lys Ala Arg Val Leu Ala Glu Ala Met Ser Gln Val Thr 720 Asn Ser Ala Thr Ile Met Met Gln Arg Gly Asn Phe Arg Asn Gln Lys 736 Lys Thr Val Lys Cys Phe Asn Cys Gly Lys Glu Gly His Ile Ala Lys 752 Asn Cys Arg Ala Ser Arg Lys Lys Arg Arg Arg Lys Lys Arg Arg Lys 768 Lys Lys 770 Met Tyr Tyr Leu Glu Phe Gln Gln Gln Gln Gln Leu Leu Asp Val Val 16 Lys Arg Gln Gln Glu Leu Leu Arg Leu Thr Val Trp Gly Thr Lys Asn 32 Leu Gln Ala Arg Val Thr Ala Ile Glu Lys Tyr Leu Gln Asp Gln Ala 48 Arg Leu Asn Ser Trp Gly Cys Ala Phe Arg Gln Val Cys His Thr Thr 64 Val Pro Trp Val Asn Asp Ser Leu Ala Pro Asp Trp Asp Asn Met Thr 80 Trp Gln Glu Trp Glu Lys Gln Val Arg Tyr Leu Glu Ala Asn Ile Ser 96 Lys Ser Leu Glu Gln Ala Gln Ile Gln Gln Glu Lys Asn Met Tyr Glu 112 Leu Gln Lys Leu Asn Ser Trp Asp Asp Pro Leu Glu Ser Cys Asp Lys 128 Cys Gln Leu Lys Gly Glu Ala Met His Gly Gln Val Asp Cys Ser Pro 144 Gly Ile Trp Gln Leu Asp Cys Thr His Leu Glu Gly Lys Ile Ile Leu 160 Val Ala Val His Val Ala Ser Gly Tyr Ile Glu Ala Glu Val Ile Pro 176 Ala Glu Thr Gly Gln Glu Thr Ala Tyr Phe Ile Leu Lys Leu Ala Gly 192 Arg Trp Pro Val Lys Val Ile His Thr Asp Asn Gly Ser Asn Phe Thr 208 Ser Thr Thr Val Lys Ala Ala Cys Trp Trp Ala Gly Ile Lys Gln Glu 224 Phe Gly Ile Pro Tyr Asn Pro Gln Ser Gln Gly Val Val Gl u Ser Met 240 Asn Lys Glu Leu Lys Lys Ile Ile Gly Gln Val Arg Asp Gln Ala Glu 256 His Leu Lys Thr Ala Val Gln Met Ala Val Phe Ile His Asn Phe Lys 272 Arg Lys Gly Gly Ile Gly Gly Tyr Ser Ala Gly Glu Arg Ile Val Asp 288 Ile Ile Ala Thr Asp Ile Gln Thr Lys Glu Leu Gln Lys Gln Ile Ile 304 Lys Ile Gln Asn Phe Arg Val Tyr Tyr Arg Asp Ser Arg Asp Pro Leu 320 Trp Lys Gly Pro Ala Lys Leu Leu Trp Lys Gly Glu Gly Ala Val Val 336 Ile Gln Asp Asn Ser Glu Ile Lys Val Val Pro Arg Arg Lys Ala Lys 352 Ile Ile Arg Asp Tyr Gly Lys Gln Ser Asp Arg Ser Ser Arg Arg Gl Gln 368 Thr Arg Gln Leu Leu Ser Gly Ile Val Gln Gln Gln Asn Asn Leu Leu 384 Arg Ala Ile Glu Thr Gln Gln His Leu Leu Gln Leu Thr Val Trp Gly 400 Ile Lys Gln Leu Gln Ala Arg Val Leu Ala Val Glu Arg Tyr Leu Gln 416 Asp Gln Arg Leu Leu Gly Ile Trp Gly Cys Ser Gly Lys Leu Ile Cys 432 Thr Thr Thr Val Pro Trp Asn Thr Ser Trp Ser Asn Lys Ser Leu Asp 448 Thr Ile Trp His Asn Met Thr Trp Met Glu Trp Glu Arg Glu Ile Asp 464 Asn Tyr Thr Ser Ser Asp Lys Gly As n Ser Ser Gln Val Ser Gln Asn 480 Tyr Pro Ile Val Gln Asn Leu Gln Gly Gln Met Val His Gln Ala Ile 496 Ser Pro Arg Thr Leu Asn Ala Trp Val Lys Val Ile Glu Glu Lys Ala 512 Phe Ser Pro Glu Val Ile Pro Met Phe Ser Ala Leu Ser Glu Gly Ala 528 Thr Pro Gln Asp Leu Asn Thr Met Leu Asn Thr Val Gly Gly His Gln 544 Ala Ala Met Gln Met Leu Lys Glu Thr Ile Asn Glu Glu Ala Ala Glu 560 Trp Asp Arg Val His Pro Val His Ala Gly Pro Ile Ala Pro Gly Gln 576 Met Arg Glu Pro Arg Gly Ser Asp Ile Ala Gly Thr Thr Ser Thr Leu 592 Gln Glu Gln Ile Gly Trp Met Thr Asn Asn Pro Pro Ile Pro Val Gly 608 Glu Ile Tyr Lys Arg Trp Ile Ile Leu Gly Leu Asn Lys Ile Val Arg 624 Met Tyr Ser Pro Val Ser Ile Leu Asp Ile Arg Gln Gly Pro Lys Glu 640 Pro Phe Arg Asp Tyr Val Asp Arg Phe Tyr Lys Thr Leu Arg Ala Glu 656 Gln Ala Ser Gln Glu Val Lys Asn Trp Met Thr Glu Thr Leu Leu Val 672 Gln Asn Ala Asn Pro Asp Cys Lys Thr Ile Leu Lys Ala Leu Gly Pro 688 Ala Ala Thr Leu Glu Glu Met Met Thr Ala Cys Gln Gly Val Gly Gly 704 Pro Gly His Ly s Ala Arg Val Leu Ala Glu Ala Met Ser Gln Val Thr 720 Asn Ser Ala Thr Ile Met Met Gln Arg Gly Asn Phe Arg Asn Gln Lys 736 Lys Thr Val Lys Cys Phe Asn Cys Gly Lys Glu Gly His Ile Ala Lys 752 Asn Cys Arg Ala Ser Arg Lys Lys Arg Arg Arg Lys Lys Arg Arg Lys 768 Lys Lys 770

Claims

P AT E N T AN S P R Ü C H E P AT ENT AT SPEECH
1. Verfahren zur enzymatischen Behandlung von Substraten, d a d u r c h g e k e n n z e i c h n e t , daß man das zu behandelnde Substrat mit einem Biokatalysator in Kontakt bringt, der erhalten wurde, indem man ein Gen, das für eine biologisch aktive Substanz kodiert und ein DNA-Fragment, das für ein Bindepeptid, das mit einem Trägermaterial in Wechselwirkung treten kann, kodiert, zur Erzeugung eines Fusionsproteins kombiniert, in einen geeigneten Vektor insertiert, in einen geeigneten Organismus transformiert, den Organismus kultiviert, die Zellen aufschließt, und das das Fusionsprotein enthaltende Lysat mit einem Trägermaterial in Kontakt bringt, das mit dem Bindepeptid bindefähig ist, wobei das Fusionsprotein an das Trägermaterial durch zwischenmolekulare Wechselwirkung bindet und anschließend das enzymatisch behandelte Substrat gewinnt. 1. A process for the enzymatic treatment of substrates, characterized in that the substrate to be treated is brought into contact with a biocatalyst obtained by using a gene coding for a biologically active substance and a DNA fragment coding for a binding peptide , which can interact with a carrier material, encodes, combines to produce a fusion protein, inserts into a suitable vector, transforms into a suitable organism, cultivates the organism, disrupts the cells, and brings the lysate containing the fusion protein into contact with a carrier material , which is capable of binding with the binding peptide, the fusion protein binding to the carrier material by intermolecular interaction and then recovering the enzymatically treated substrate.
2. Verfahren nach Anspruch 1, d a d u r c h 2. The method according to claim 1, d a d u r c h
g e k e n n z e i c h n e t , daß man als Bindepeptid ein Peptid verwendet, das überwiegend aus positiv geladenen Aminosäuren aufgebaut ist.  It is important that a peptide is used as the binding peptide which is predominantly composed of positively charged amino acids.
3. Verfahren nach Anspruch 2, d a d u r c h 3. The method according to claim 2, d a d u r c h
g e k e n n z e i c h n e t , daß man als Bindepeptid ein Peptid verwendet, das überwiegend aus den Aminosäuren Lysin und/oder Arginin aufgebaut ist.  It is a fact that a peptide is used as the binding peptide which is predominantly composed of the amino acids lysine and / or arginine.
4. Verfahren nach Anspruch 1, d a d u r c h 4. The method according to claim 1, d a d u r c h
g e k e n n z e i c h n e t , daß man als Bindepeptid ein Peptid verwendet, das überwiegend aus negativ geladenen Aminosäuren aufgebaut ist. characterized in that a peptide is used as the binding peptide, which is predominantly composed of negatively charged amino acids.
5. Verfahren nach Anspruch 4, d a d u r c h 5. The method according to claim 4, d a d u r c h
g e k e n n z e i c h n e t , daß man als Bindepeptid ein Peptid verwendet, das überwiegend aus den Aminosäuren Asparaginsäure und/oder Glutaminsäure aufgebaut ist.  It is a fact that the binding peptide used is a peptide which is predominantly composed of the amino acids aspartic acid and / or glutamic acid.
6. Verfahren nach Anspruch 1, d a d u r c h 6. The method according to claim 1, d a d u r c h
g e k e n n z e i c h n e t , daß man als Bindepeptid ein Peptid verwendet, das überwiegend aus der Aminosäure Histidin aufgebaut ist.  It is a fact that the binding peptide used is a peptide which is predominantly composed of the amino acid histidine.
7. Verfahren nach Anspruch 1, d a d u r c h 7. The method according to claim 1, d a d u r c h
g e k e n n z e i c h n e t , daß man als Bindepeptid ein Peptid verwendet, das überwiegend aus den Aminosäuren Alanin, Valin, Leucin, Isoleucin, Phenylalanin, Tryptophan und/oder Tyrosin aufgebaut ist.  It is a requirement that a peptide is used as the binding peptide which is predominantly composed of the amino acids alanine, valine, leucine, isoleucine, phenylalanine, tryptophan and / or tyrosine.
8. Verfahren nach einem der vorhergehenden Ansprüche, 8. The method according to any one of the preceding claims,
d a d u r c h g e k e n n z e i c h n e t , daß das Bindepeptid zusätzlich noch an der Verknüpfungsstelle mit der biologisch aktiven Substanz einen Spacer aus 1 bis 10 Aminosäureresten aufweist.  that the binding peptide additionally has a spacer of 1 to 10 amino acid residues at the point of attachment to the biologically active substance.
9. Verfahren nach Anspruch 8, d a d u r c h 9. The method according to claim 8, d a d u r c h
g e k e n n z e i c h n e t , daß der Spacer 2 bis 4 Aminosäurereste aufweist.  it is that the spacer has 2 to 4 amino acid residues.
10. Verfahren nach Anspruch 8 oder 9, d a d u r c h 10. The method according to claim 8 or 9, d a d u r c h
g e k e n n z e i c h n e t , daß für den Spacer die Aminosäuren Prolin, Glycin, Alanin und/oder Serin verwendet werden.  it is that the amino acids proline, glycine, alanine and / or serine are used for the spacer.
11. Verfahren nach einem der vorhergehenden Ansprüche, 11. The method according to any one of the preceding claims,
d a d u r c h g e k e n n z e i c h n e t , daß das Ende des Fusionsproteins, das das Bindepeptid trägt, durch eine anfusionierte, nicht geladene Aminosäure (vorzugsweise Prolin) geschützt wird. characterized in that the end of the fusion protein carrying the binding peptide is protected by a fused, uncharged amino acid (preferably proline).
12. Verfahren nach Anspruch 11, d a d u r c h 12. The method according to claim 11, d a d u r c h
g e k e n n z e i c h n e t , daß als schützende Aminosäure Prolin verwendet wird.  g e n e n c e i c h n e t that proline is used as a protective amino acid.
13. Verfahren nach einem der vorhergehenden Ansprüche, 13. The method according to any one of the preceding claims,
d a d u r c h g e k e n n z e i c h n e t , daß man als Trägermaterial ein Material verwendet, das negativ geladene Gruppen aufweist.  d a d u r c h g e k e n n z e i c h n e t that a material is used as the carrier material which has negatively charged groups.
14. Verfahren nach einem der Ansprüche 1 bis 12, 14. The method according to any one of claims 1 to 12,
d a d u r c h g e k e n n z e i c h n e t , daß man als Trägermaterial ein Material verwendet, das positiv geladene Gruppen aufweist.  d a d u r c h g e k e n n z e i c h n e t that a material is used as the carrier material which has positively charged groups.
15. Verfahren nach einem der Ansprüche 1 bis 12, 15. The method according to any one of claims 1 to 12,
d a d u r c h g e k e n n z e i c h n e t , daß man als Trägermaterial ein Material verwendet, das komplexierte Metallionen aufweist.  d a d u r c h g e k e n n z e i c h n e t that a material is used as the carrier material which has complexed metal ions.
16. Verfahren nach einem der Ansprüche 1 bis 12, 16. The method according to any one of claims 1 to 12,
d a d u r c h g e k e n n z e i c h n e t , daß man als Trägermaterial ein Material verwendet, das hydrophobe Gruppen aufweist.  d a d u r c h g e k e n n z e i c h n e t that a material is used as the carrier material which has hydrophobic groups.
17. Verfahren zum Nachweis von spezifisch bindefähigen Substanzen nach dem Prinzip des heterogenen Immunoassays unter Verwendung eines Festphasen-gebundenen Rezeptors, d a d u r c h g e k e n n z e i c h n e t , daß man die Probelösung sowie mindestens einen markierten, mit der nachzuweisenden Substanz bindefähigen Rezeptor in Kontakt bringt mit einem Festphasen-gebundenen Rezeptor, der erhalten wurde, indem man ein Gen, das für eine immunologisch aktive Substanz kodiert und ein DNA- Fragment, das für ein Bindepeptid, das mit einem Trägermaterial in Wechselwirkung treten kann, kodiert, zur Erzeugung eines Fusionsproteins kombiniert, in einen geeigneten Vektor insertiert, in einen geeigneten Organismus transformiert, den Organismus kultiviert, die Zellen aufschließt und das das Fusionsprotein enthaltende Lysat mit einem Trägermaterial in Kontakt bringt, das mit dem Bindepeptid bindefähig ist, wobei das Fusionsprotein an das Trägermaterial durch zwischenmolekulare Wechselwirkung bindet und nach Inkubation die feste von der flüssigen Phase trennt und die Markierung in einer der beiden Phasen bestimmt. 17. A method for the detection of specifically bindable substances according to the principle of heterogeneous immunoassay using a solid phase bound receptor, characterized in that the sample solution and at least one labeled receptor which is bindable with the substance to be detected are brought into contact with a solid phase bound receptor, which has been obtained by using a gene which codes for an immunologically active substance and a DNA fragment which codes for a binding peptide which can interact with a carrier material Generation of a fusion protein is combined, inserted into a suitable vector, transformed into a suitable organism, the organism is cultivated, the cells are disrupted and the lysate containing the fusion protein is brought into contact with a carrier material which is capable of binding to the binding peptide, the fusion protein being attached to the carrier material binds through intermolecular interaction and, after incubation, separates the solid from the liquid phase and determines the labeling in one of the two phases.
18. Verfahren zum Nachweis von spezifisch bindefähigen Substanzen nach dem Prinzip des homogenen Immunoassays unter Verwendung eines Partikel-gebundenen Rezeptors, d a d u r c h g e k e n n z e i c h n e t , daß man die Probe mindestens mit einem mit der nachzuweisenden Substanz bindefähigen Partikel-gebundenen Rezeptor inkubiert, der erhalten wurde, indem man ein Gen das für eine immunologisch aktive Substanz kodiert und ein DNA-Fragment, das für ein Bindepeptid, das mit einem Trägermaterial in Wechselwirkung treten kann, kodiert, zur Erzeugung eines Fusionsproteins kombiniert, in einen geeigneten Vektor insertiert, in einen geeigneten Organismus transformiert, den Organismus kultiviert, die Zellen aufschließt und das das Fusionsprotein enthaltende Lysat mit einem Trägermaterial in Kontakt bringt, das mit dem Bindepeptid bindefähig ist, wobei das Fusionsprotein an das Trägermaterial durch zwischenmolekulare Wechselwirkung bindet und nach Inkubation die feste von der flüssigen Phase trennt und nach Inkubation die Agglutination bestimmt. 18. A method for the detection of specifically bindable substances according to the principle of homogeneous immunoassay using a particle-bound receptor, characterized in that the sample is incubated with at least one particle-bound receptor which is bindable with the substance to be detected and which was obtained by using a The gene coding for an immunologically active substance and a DNA fragment coding for a binding peptide which can interact with a carrier material, combined to produce a fusion protein, inserted into a suitable vector, transformed into a suitable organism, the organism cultured, disrupting the cells and bringing the fusion protein-containing lysate into contact with a carrier material which is capable of binding to the binding peptide, the fusion protein binding to the carrier material by intermolecular interaction and, after incubation, the solid from the liquid phase separates and agglutination is determined after incubation.
PCT/EP1991/000086 1990-01-19 1991-01-18 Process for enzymatically treating substrates WO1991010910A2 (en)

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FI914389A FI914389A0 (en) 1990-01-19 1991-09-18 FOERFARANDE FOER ENZYMATISK BEHANDLING AV SUBSTRAT.
KR1019910701147A KR920701822A (en) 1990-01-19 1991-09-18 Method of enzymatic treatment of substrate
NO91913673A NO913673L (en) 1990-01-19 1991-09-18 PROCEDURE FOR ENZYMATIC TREATMENT OF SUBSTRATES.

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DEP4001508.4 1990-01-19
DE4001508A DE4001508A1 (en) 1990-01-19 1990-01-19 Treating substrate with immobilised enzyme as fusion protein - with binding peptide specifically interacting with carrier, and new immunoassay using receptor-binding peptide fusion protein
DE4002636A DE4002636A1 (en) 1990-01-30 1990-01-30 EXPRESSION OF HIV1 AND 2 POLYPEPTIDES AND THEIR USE
DEP4002636.1 1990-01-30

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EP0645454A2 (en) * 1993-06-22 1995-03-29 Vladimir Glebovich Lunin Chimeric somatostatin containing protein and coding DNA, immunogenic compositions and method for increasing farm animal productivity
EP0645454A3 (en) * 1993-06-22 1997-01-15 Vladimir Glebovich Lunin Chimeric somatostatin containing protein and coding DNA, immunogenic compositions and method for increasing farm animal productivity.

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EP0464184A1 (en) 1992-01-08
AU7072491A (en) 1991-08-05
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KR920701822A (en) 1992-08-12
AU633686B2 (en) 1993-02-04
JPH04503610A (en) 1992-07-02

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