EP0464184A1 - Procede de traitement enzymatique de substrats - Google Patents

Procede de traitement enzymatique de substrats

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Publication number
EP0464184A1
EP0464184A1 EP91903190A EP91903190A EP0464184A1 EP 0464184 A1 EP0464184 A1 EP 0464184A1 EP 91903190 A EP91903190 A EP 91903190A EP 91903190 A EP91903190 A EP 91903190A EP 0464184 A1 EP0464184 A1 EP 0464184A1
Authority
EP
European Patent Office
Prior art keywords
fusion protein
carrier material
binding peptide
binding
peptide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP91903190A
Other languages
German (de)
English (en)
Inventor
Rainer Dr. Rudolph
Erhard Dr. Kopetzki
Stephan Dr. Fischer
Adelbert Dr. Grossmann
Bärbel HÖLL-NEUGEBAUER
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Roche Diagnostics GmbH
Original Assignee
Roche Diagnostics GmbH
Boehringer Mannheim GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from DE4001508A external-priority patent/DE4001508A1/de
Priority claimed from DE4002636A external-priority patent/DE4002636A1/de
Application filed by Roche Diagnostics GmbH, Boehringer Mannheim GmbH filed Critical Roche Diagnostics GmbH
Publication of EP0464184A1 publication Critical patent/EP0464184A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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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  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Cell Biology (AREA)
  • Organic Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Virology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • AIDS & HIV (AREA)
  • Plant Pathology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Peptides Or Proteins (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

Afin de soumettre des substrats à un traitement enzymatique, on met le substrat à traiter en contact avec un biocatalyseur obtenu en combinant un gène de codage pour une substance biologiquement active avec un fragment d'ADN de codage d'un peptide de liaison capable d'interaction avec un matériau de support, de manière à obtenir une protéine de fusion. On insère ensuite celle-ci dans un vecteur approprié, on la transforme dans un organisme approprié, on met en culture ledit organisme, on en dissout les cellules et on met le lysat contenant la protéine de fusion en contact avec un matériau de support capable de se lier au peptide de liaison. La protéine de fusion se lie au matériau de support par interaction intermoléculaire, ce qui permet d'obtenir le substrat enzymatiquement traité.
EP91903190A 1990-01-19 1991-01-18 Procede de traitement enzymatique de substrats Withdrawn EP0464184A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
DE4001508 1990-01-19
DE4001508A DE4001508A1 (de) 1990-01-19 1990-01-19 Verfahren zur enzymatischen behandlung von substraten
DE4002636A DE4002636A1 (de) 1990-01-30 1990-01-30 Expression von hiv1- und 2-polypeptiden und deren verwendung
DE4002636 1990-01-30

Publications (1)

Publication Number Publication Date
EP0464184A1 true EP0464184A1 (fr) 1992-01-08

Family

ID=25889228

Family Applications (1)

Application Number Title Priority Date Filing Date
EP91903190A Withdrawn EP0464184A1 (fr) 1990-01-19 1991-01-18 Procede de traitement enzymatique de substrats

Country Status (6)

Country Link
EP (1) EP0464184A1 (fr)
JP (1) JPH04503610A (fr)
KR (1) KR920701822A (fr)
AU (1) AU633686B2 (fr)
CA (1) CA2047235A1 (fr)
WO (1) WO1991010910A2 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6316004B1 (en) * 1993-06-22 2001-11-13 T. Tikhonenko Chimeric somatostatin containing protein and encoding DNA, plasmids of expression, method for preparing chimeric protein, strain-producers, immunogenic composition, method for increasing the productivity of farm animals

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4532207A (en) * 1982-03-19 1985-07-30 G. D. Searle & Co. Process for the preparation of polypeptides utilizing a charged amino acid polymer and exopeptidase
ATE135403T1 (de) * 1987-03-10 1996-03-15 New England Biolabs Inc Herstellung und reinigung eines proteins, das mit einem bindungsprotein fusioniert ist
CA1340522C (fr) * 1987-03-10 1999-05-04 Heinz Dobeli Proteins hybrides renfermant des histidines voisines pour une purification amelioree
US4921787A (en) * 1987-05-01 1990-05-01 Cambridge Bioscience Corporation Detection of antibodies to human immunodeficiency virus by agglutination of antigen coated latex
AU609241B2 (en) * 1988-01-29 1991-04-26 Abbott Laboratories Ion-capture assays and devices
DE58905660D1 (de) * 1988-04-25 1993-10-28 Hoffmann La Roche Diagnose-Hilfsmittel.
DE3822045A1 (de) * 1988-06-30 1990-01-11 Hoechst Ag Verfahren zur herstellung von proteinen

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9110910A2 *

Also Published As

Publication number Publication date
WO1991010910A3 (fr) 1991-10-03
AU7072491A (en) 1991-08-05
JPH04503610A (ja) 1992-07-02
WO1991010910A2 (fr) 1991-07-25
AU633686B2 (en) 1993-02-04
CA2047235A1 (fr) 1991-07-20
KR920701822A (ko) 1992-08-12

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