EP1001992A2 - Procede pour la fixation, catalysee par la transglutaminase, d'une proteine ou d'un peptide sur un support - Google Patents

Procede pour la fixation, catalysee par la transglutaminase, d'une proteine ou d'un peptide sur un support

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Publication number
EP1001992A2
EP1001992A2 EP98945120A EP98945120A EP1001992A2 EP 1001992 A2 EP1001992 A2 EP 1001992A2 EP 98945120 A EP98945120 A EP 98945120A EP 98945120 A EP98945120 A EP 98945120A EP 1001992 A2 EP1001992 A2 EP 1001992A2
Authority
EP
European Patent Office
Prior art keywords
protein
carrier
transglutaminase
peptide
coupled
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Withdrawn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
EP98945120A
Other languages
German (de)
English (en)
Inventor
Hans-Lothar Fuchsbauer
Ralf Pasternack
Rolf Eymann
Jens Otterbach
Uwe Bechtold
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.)
N Zyme Biotec GmbH
Original Assignee
Fuchsbauer Hans-Lothar
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
Application filed by Fuchsbauer Hans-Lothar filed Critical Fuchsbauer Hans-Lothar
Publication of EP1001992A2 publication Critical patent/EP1001992A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C07K17/02—Peptides being immobilised on, or in, an organic carrier
    • 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
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C12Q1/52—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving transaminase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/91045—Acyltransferases (2.3)
    • G01N2333/91074—Aminoacyltransferases (general) (2.3.2)
    • G01N2333/9108—Aminoacyltransferases (general) (2.3.2) with definite EC number (2.3.2.-)
    • G01N2333/91085—Transglutaminases; Factor XIIIq (2.3.2.13)

Definitions

  • the present invention relates to a method for transglutaminase-catalyzed coupling of protein or peptide to a carrier while maintaining at least 50% of the biological activity of the protein or peptide, carrier-coupled protein immobilizates and carrier-protein conjugates obtainable according to this method, and the use of carrier-coupled protein immobilizates and carrier-protein conjugates according to the invention.
  • the covalent linking of proteins to stable aggregates or their covalent binding to carrier materials are important processes in medical technology, food technology and diagnostics.
  • Crosslinking usually takes place with chemical reagents, among which glutardialdehyde plays an outstanding role.
  • Other organic compounds with at least two reactive functional groups can also be used.
  • Stable binding can also be achieved by chemically activating a protein before coupling it with a second protein.
  • a common method is, for example, the oxidation of glycoproteins with periodic acid while opening a sugar ring and generating reactive carbonyl functions.
  • the carrier material In immobilization processes, the carrier material usually already has a suitable reactive group for the formation of a covalent bond between the protein and the carrier matrix, e.g. an oxirane group.
  • transglutaminases protein glutamine: amine - ⁇ - glutamyl transferase E.C. 2.3.2.13 specifically catalyze the construction of stable cross bridges between proteins.
  • the ⁇ -carboxamide function of glutamine side chains is transferred to the ⁇ -amino group of lysine residues with the release of ammonium ions (Folk and Finlayso ⁇ , Adv. Protein Chem. 31, 1-133 (1977)).
  • the newly formed isopeptide bond also withstands hydrolysis by proteases and is only physiologically cleaved by a ⁇ -glutamylamine cyclotransferase after the proteins have completely broken down (Fink et al., Proc. Natl. Acad. Sci. USA 77, 4564-4568 (1980 )).
  • JP 59 66 886 describes a method in which carrier material and protein to be coupled are simultaneously precipitated for the production of protein immobilized products (simultaneous immobilization process).
  • various enzymes glucose oxidase, catalase, diaphorase, RNase, glucosidase, mannosidase, glutamate dehydrogenase
  • This method has also been successfully used to produce active casein membranes (JP 61 227 783 and Motoki et al., Agric. Biol. Chem.
  • the object of the invention is therefore to provide a method with which proteins or peptides can be coupled to a carrier while maintaining their biological activity.
  • this object is achieved by a process for the transglutaminase-catalyzed coupling of protein or peptide to a carrier while maintaining at least 50% of the biological activity of the protein or peptide, the carrier being a bioactive molecule or a predetermined insoluble one Is a matrix comprising the step of bringing the carrier into contact with the protein to be coupled and with transglutaminase, the carrier and the protein or peptide acting as an acyl and / or amine donor and being suitable as a transglutaminase substrate.
  • biological activity of the protein or peptide means the respective enzymatic activity of enzymes, the antibody-binding activity of antibodies, the inhibitory activity of inhibitors and the antigenicity of antigens.
  • biological activity not only whole proteins, but also protein fragments or peptides can be biologically active.
  • the method according to the invention enables protein or peptide immobilizates and conjugates to be produced with all carriers accepted by a transglutaminase.
  • proteins and peptides require glutamine and / or lysine residues that are accessible to transglutaminase.
  • Other compounds and modified proteins or peptides must have at least one acid amide function or a primary amine group.
  • Proteins and other compounds are also suitable as starting materials for the enzymatic crosslinking if they have been modified beforehand by extension with an oligo or polyglutaminyl chain, a glutaminyl peptide, an oligo or polylysine chain or a primary amine with the aid of chemical and / or molecular biological methods have been.
  • transglutaminase substrate a suitable enzyme substrate or reaction partners
  • the suitability as a transglutaminase substrate can be determined by the incorporation of usually fluorescent transglutaminase substrates. the.
  • acyl and glutamine donors on the one hand and amine donors (glutamine acceptors ⁇ ) on the other.
  • example 2 With proteins, two different incubation approaches should be carried out for each protein (example 2).
  • a labeled amino e.g. C-DNA (I)
  • the other a labeled acid amide e.g. CBZ-Gln-Gly-C-DNA (II)
  • reactive lysine side chains e.g. CBZ-Gln-Gly-C-DNA (II)
  • the proteins are separated electrophoretically and then visible by irradiation with UV light of wavelength 366 ⁇ m. makes.
  • Peptides and other small molecules can be separated from the excess reagent by chromatography, for example analytical HPLC, and can be recognized as a transglutaminase substrate by their emission.
  • radioactive or biotinylated labeling reagents eg Biotinylamidopentylamine from Pierce
  • Successful labeling is then detected after separation by autoradiography or with avidin / streptavidin-enzyme conjugates.
  • Unmodified proteins can be classified into four categories:
  • Table 1 shows examples of important bioactive proteins with reactive side chains that can serve as transglutaminase substrates. In principle, other biopolymers and synthetic polymers can be recognized as transglutaminase substrates with the test method described. TABLE 1
  • the method according to the invention can be carried out with any type of acyl- and / or ami-donor, provided that this is suitable as a transglutaminase substrate, which, as stated above, can be tested.
  • the protein or peptide to be coupled is a glutamine donor and the carrier comprises reactive amine groups.
  • the protein or peptide to be coupled is a glutamine acceptor and the carrier provides reactive carboxamide groups.
  • the carrier can contain amine groups or carboxamide groups or amine and carboxamide groups.
  • the carrier can also be a protein, so that the reactive amine groups of the carrier also include, for example, lysine side chains reactive carboxamide groups of the support can be reactive glutamine side chains.
  • the carrier can be a bioactive molecule in solution.
  • the bioactive molecule is preferably an enzyme.
  • the coupling of a protein or peptide to an enzyme is used, for example, to provide molecules with two different enzymatic activities.
  • the crosslinking of two molecules can also be useful for the provision of protein dimers or higher forms of aggregation which, for example, have a biological activity as a ligand of a receptor.
  • reactive amines must be provided by at least one reaction partner and reactive carboxamide groups by at least one further reaction partner.
  • the carrier is a predetermined insoluble matrix.
  • proteins such as gelatin and casein
  • Cross-linked proteins are preferred as the insoluble matrix.
  • the protein or peptide to be coupled can be a native protein or peptide, a recombinant or a synthetically produced one. Furthermore, the protein or peptide to be coupled can be modified or unmodified. Modifications mean mutations of the primary structure including deletions and insertions as well as amino acid exchanges, as well as chemical modifications such as the subsequent PEGylation, glycosylation or deglycosylation, the subsequent addition or insertion of cysteine residues etc. The same applies to the wearer and refers to both the insoluble carrier matrix and the bioactive molecule. According to the invention, the protein or peptide to be coupled retains at least 50% of the initial activity after it has been coupled. In preferred embodiments, however, the activity is 60% or 70%, particularly preferably 80% or 90% or even more than 90% of its starting activity.
  • the biological activity of the bioactive molecule also remains at least 50%.
  • Preferred embodiments also provide activities of 60 or 70%, preferably 80, 90 or more than 90%.
  • the method according to the invention allows the use of any protein glutamine: amine ⁇ -glutamyl transferase.
  • Bacterial transglutaminase, plant or mammalian transglutaminase are preferred.
  • Transglutaminases have also been described in mollusks and crustaceans and are likely to be ubiquitous.
  • the use of a bacterial transglutaminase is particularly preferred.
  • the best characterized bacterial transglutaminase is the transglutaminase from Streptoverticillium mobaraense.
  • the transglutaminase from guinea pig liver is preferably used.
  • An example of a mammalian transglutaminase with high substrate specificity is the blood coagulation factor XII l a .
  • the conditions for performing the transglutaminase-catalyzed coupling depend on the origin of the transglutaminase used.
  • a carrier for example an enzymatically crosslinked gelatin film (example 3)
  • the temperatures during the enzymatic linkage are preferably 25 to 55 ° C. and particularly preferably 30 to 45 ° C.
  • the reaction is at 37 ° C or 37 ° C + 5 ° C.
  • the incubation time is preferably between half a and 18 hours and is particularly preferably between 1 and 4 hours.
  • the pH of the buffered solution should be between pH 5 and 9; preferred ranges depend on the buffer system used and for most buffers are pH 6 to 8, particularly preferably pH 7 + . 0.5.
  • the following buffer solutions can be used as buffers:
  • Phosphate buffer pH 6.0 - 8.0, preferably pH 6.0 - 7.0
  • TRIS 1 -HCI buffer, pH 7.0 - 9.0, preferably pH 7.0 - 8.5
  • TRIS acetate pH 6.0 - 9.0, preferably pH 6.0
  • Tricin 2 -HCI buffer, pH 7.0 - 9.0, preferably pH 7.5
  • MOPS 3 buffer pH 6.0 - 8.0, preferably pH 7.5
  • TEA 4 buffer, pH 6.0 - 9.0, preferably pH 7.5
  • the preferred buffer is tricin-HCl buffer
  • the mammalian enzyme obtained from guinea pig liver gives the best results in TRIS-HCl buffer with EDTA, dithiotreitol and calcium ions.
  • the carrier-coupled protein immobilizates obtained have an unexpectedly high document density.
  • a gelatin protein A is obtained. Immobilisate with a coverage of at least 2 ng (50 fmol) protein A per mm 2 foil, if 1 ⁇ mol protein A and 2 g (approx. 4 dm 2 ) gelatin foil in Tris-acetate buffer, pH 6.0, with 50 nmol bacterial transglutaminase are incubated for 60 min at 37 ° C (Examples 4 and 5).
  • immobilized protein A still binds the maximum number of 2 antibody molecules of the IgG class. However, a higher document density may be achieved with this procedure.
  • the protein or peptide to be coupled can comprise more than one protein and / or peptide sequence.
  • several proteins or biologically active peptides can be immobilized in a very small space (multi-enzyme immobilization).
  • the optimal molar ratio of protein or peptide to carrier to be coupled can be determined experimentally according to the respective needs.
  • the mixing ratio for both compounds depends on the number of reactive groups, the molecular size and the intended use of the conjugate.
  • the molar ratio is usually 5: 1 to 1: 100 (protein or peptide to carrier to be coupled).
  • Preferred ranges are 1: 5 to 1:50 in the case of coupling to an insoluble support.
  • transglutamine substrate molecules ideally a glutamine donor (category 1) and a glutamine acceptor (category 2), are incubated with transglutaminase at pH 5-9 and temperatures of 20-60 ° C.
  • glutamine donor category 1
  • glutamine acceptor category 2
  • the preferred ratio of protein or peptide to be coupled to bioactive molecule is 1:20 to 5: 1.
  • the product can be purified if necessary by dialysis, ultrafiltration, precipitation or a chromatographic process.
  • One of the advantages of the method according to the invention is that the biological activity of the reactants is generally completely retained.
  • a further decisive advantage of the process according to the invention over a known chemical coupling can be seen above all in the fact that the expensive, unreacted starting compounds can be recovered by separation processes.
  • a conjugate with two different biological activities can be produced by crosslinking protein G (a category 2 protein, see also Table 3) with soy peroxidase (a category 3 protein) in a 1:20 ratio with transglutaminase (example 7).
  • Successful coupling can be checked by fixing antibodies of class G (IgG) that specifically bind protein G to a nitrocellulose membrane, applying the test solution and staining with 4-chloro-1-naphthol and hydrogen peroxide after several washing steps ("Dot - Blot ").
  • the conjugates formed are quantified using a microtiter plate assay (Example 8). Enrichment or complete purification of the protein G-peroxidase conjugates and recovery of the unreacted starting materials is achieved by chromatography on an anion exchanger (example 9) or by geipermeation chromatography.
  • the invention further relates to a carrier-coupled protein immobilizate and to a carrier-protein conjugate which can be obtained by the method according to the invention.
  • a preferred carrier-protein conjugate is a transglutaminase-catalyzed coupling obtained conjugate of monoclonal or polyclonal antibody and marker enzyme.
  • This marker enzyme can be, for example, alkaline phosphatase, horseradish peroxidase or any other marker enzyme known in the art.
  • the transglutamine-catalyzed coupling allows the connection of an antibody molecule with up to 20 enzyme molecules, which amplifies the signal, for example in subsequent ones Antigen-antibody reactions allowed. Oligomeric enzymes created by cross-linking also amplify the signal.
  • the invention also relates to the use of a carrier-coupled protein immobilizate or carrier-protein conjugate in the enzymatic analysis.
  • the substrate proteins listed in Table 3 can be coupled to an insoluble support and used for establishing a new transglutaminase activity assays, in particular for the development of a specific and sensitive analytical method for the determination of active Factor XIII a in human blood serum.
  • a microtiter plate is coated with a good transglutaminase substrate, eg casein. Binding sites that were still free were saturated with bovine serum albumin. After adding an enzyme that has reactive glutamine or lysine residues, e.g.
  • the transglutaminase sample or a comparison sample is pipetted in and incubated at pH 5 for 0.5 to 5 hours or longer, depending on the desired sensitivity to 9 and temperatures of 20-50 ° C.
  • the covalently bound enzyme fraction is quantitatively determined using a colorimetric enzyme reaction, for example in the case of alkaline phosphatase via the hydrolysis of para-nitrophenyl phosphate. If the substrate proteins protein A and protein G, avidin and streptavidin are used, a second incubation with antibody-enzyme conjugates or biotin-enzyme conjugates is necessary before a detection reaction can be carried out. This improves the sensitivity through a higher enzyme concentration on the microtiter plate, since protein A and protein G can bind up to 2, avidin and streptavidin up to 4 conjugate molecules.
  • a modification of the test can be that the coated microtiter plate is replaced by a cross-linked gelatin foil. This is then immersed in an enzyme solution, to which the transglutaminase sample is subsequently added becomes. After incubation and multiple washing, the gelatin strip can be used directly for the photometric measurement in a cuvette.
  • multi-enzyme immobilizates can be produced by sequential immobilization on supports, which can be used, for example, in a multi-stage synthesis of organic or biological compounds.
  • the multi-enzyme immobilizates prepared according to the invention are also suitable for diagnostic applications, for example for the determination of glycerol lipids after enzymatic release and oxidation of glycerol, and photometric measurement of the reduction equivalents formed in this reaction.
  • the protein immobilizates and carrier-protein conjugates according to the invention can also be used to produce biosensors.
  • Biosensor technology requires stable bioactive compounds that are fixed in the smallest possible space.
  • Active protein foils or other active insoluble protein complexes could offer advantages over soluble enzymes.
  • a biosensor could be constructed, the surface of which is coated with a specific antigen, for example hepatitis B surface virus protein. After the biosensor is brought into contact with a sample containing an unknown concentration of antibodies against HbsAg and a known concentration of a ⁇ ti-HbsAg-antibody-enzyme conjugate, the enzyme-conjugated antibodies compete with the antibodies to be determined for the binding sites.
  • the carrier-coupled protein immobilizates can be coated microtiter plates with biological activity. Active membranes, enzyme carriers and analytical test strips can also be produced using the protein immobilizates according to the invention. A large number of test strips are known in diagnostics and analysis, which are based on the activity of an enzyme immobilized on the test strip. Test strips are also increasingly used in the home. For example, a test stick was developed in Japan which, by enzymatically determining the proportion of carcassin on the surface of fish, allows a statement to be made about its freshness.
  • Figure 3 the determination of transglutaminase by covalent binding of alkaline phosphatase to a casein coated microtiter plate and subsequent hydrolysis of paranitrophenyl phosphate
  • Figure 4 shows the enrichment of protein G peroxidase conjugates by ion exchange chromatography on Fractogel EDM-TMAE 650 S.
  • Transglutaminase was derived from the culture supernatant of Streptoverticillium mobaraense as described by Gerber et al., Biochem. J. 299, 825-829 (1994), purified by chromatography on a strongly acidic ion exchanger and material. Polyacrylamide gel electrophoresis showed the enzyme as a single band with a specific activity greater than 30 U / mg when it was in accordance with that of Grossowicz et al., J. Biol. Chem. 187, 111-125 (1950), described method for hydroxamate formation with CBZ-glutaminylglycine was tested. Protein concentrations were determined using a standard bicinchoninic acid protocol as described by the manufacturer.
  • the protein solutions of the transglutaminase substrates described here are dissolved in the concentrations given in Tab. 2 in 0.02 M sodium phosphate buffer, pH 7.3.
  • Transglutaminase is obtained, for example, as described in Example 1 by cultivating Streptoverticillium mobaraense (DSM 40847) and enriched by ion exchange chromatography using a standard method (Gerber et al, Biochem. J. 299, 825-829 (1994)). The enzyme activity is determined according to the method of Grossowicz et al. (J. Biol. Chem. 187, 111-125 (1950)). The amount of enzyme used is given in Table 2.
  • the reagent solutions are mixed in the order of Table 3 and analyzed after a given incubation period.
  • Example 4 Production of a gelatin film with protein A activity
  • a crosslinked gelatin film (2 g dry matter) is heated to 37 ° C. in 70 ml 0.2 M Tris acetate buffer, pH 6.0. After adding 10 ml of 10 mM protein A in 0.2 M Tris acetate buffer, pH 6.0, and 20 ml of transglutaminase with activity 4 U / ml, the mixture is incubated at 37 ° C. for 60 min. The film is then washed twice with 100 ml of Tris acetate buffer, pH 6.0, air-dried and cut into strips (approx. 2 mg).
  • a rabbit anti-chicken antibody (IgG) conjugated to alkaline phosphatase (Sigma) is mixed with 0.05 M Tris-HCl buffer, pH 8.0, which is additionally 150 mM NaCI, 0.005% (v / v) Tween 20 and contains 0.09% (w / v) NaN 3 (AK binding buffer), diluted 1: 2000 to 1: 10000. With each dilution, a color reaction is carried out according to the scheme in Table 4. The calibration line is shown in Fig.1.
  • a 2 mg strip of gelatin protein A immobilizate is placed in AK binding buffer for 30 minutes to swell.
  • the well-drained aliquot is transferred to 100 ⁇ l of a 1: 2000 dilution of the antibody-alkaline-phosphatase conjugate and incubated at 37 ° C. for 10 min.
  • the color reaction is carried out according to the scheme in Table 4.
  • a crosslinked gelatin film without protein A is used for the comparison approach.
  • Fig. 2 shows the dependence of the protein A covering density on the concentration of the crosslinking enzyme.
  • 250 ⁇ l of a casein solution (1 mg / ml in coating buffer consisting of 40 mM Tris-HCl with 150 mM NaCl, pH 8.3) are pipetted into each well of a microtiter plate made of polystyrene.
  • the microtiter plates are kept at 4 ° C overnight incubated and remaining binding sites saturated with 250 ⁇ l of a 1% (w / v) bovine serum albumin solution in coating buffer.
  • Soybean peroxidase and protein G are dissolved in a molar ratio of 20: 1 in 0.3 M Tris-HCl buffer, pH 7.0. After adding transglutaminase (0.5 U / mg protein), the mixture is incubated at 37 ° C. for 18 h.
  • Example 8 Quantitative detection of a protein G-peroxidase conjugate
  • coating buffer consisting of 40 mM Tris-HCl with 150 mM NaCl, pH 8.3
  • a sample or reference sample eg conjugate mixture without transglutaminase
  • 100 ⁇ l of a sample or reference sample are pipetted into each well and incubated for 60 min at room temperature. It is then rinsed again three times with 200 ⁇ l of coating buffer.
  • a substrate solution is prepared from 2 ml of 0.1 M phosphate buffer, pH 6.0, 2 ml of a 5% (w / v) pyrogallol solution in H 2 O, 1 ml of 0.05% H 2 O 2 solution and 13 ml H 2 O. 100 ⁇ l of the substrate solution are pipetted into each well of the microtiter plate, and after 30 min the absorbance is measured at 405 nm.
  • Example 9 Enrichment of a protein-G-peroxidase conjugate and recovery of the starting materials
  • a 10x150 mm ion exchange column filled with Fractogel-EMD-TMAE-650 S (Merck) is equilibrated with five times the bed volume of 50 mM sodium phosphate buffer, pH 7.0. 1 ml of a protein-G-peroxidase conjugate mixture is pumped onto the column and the non-binding proteins (transglutaminase and protein G in this order) are rinsed off the column with equilibration buffer. The conjugates are then eluted within 60 min by linearly increasing the ion concentration with NaCl from 0 to 0.15 M. The highest conjugate concentrations are found in fractions at 0.08-0.12 M NaCI (Fig. 4).

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Abstract

L'invention concerne un procédé pour la fixation, catalysée par la transglutaminase, d'une protéine ou d'un peptide sur un support, tout en conservant au moins 50 % de l'activité biologique de la protéine ou du peptide, des protéines fixées au support immobilisées et des supports-protéines conjugués obtenus selon ce procédé, ainsi que l'utilisation des protéines fixées au support immobilisées et des supports-protéines conjugués conformes à l'invention. Il est déjà connu d'immobiliser simultanément des enzymes avec des matériaux supports, par fixation catalysée par la transglutaminase. Ce procédé n'est toutefois pas approprié pour des enzymes avec des molécules substrats. Des essais poussés visant à lier des enzymes avec des substrats polymères sur des matériaux échangeurs d'ions et à les stabiliser avec la transglutaminase n'ont conduit qu'à des produits immobilisés de très faible activité. Le nouveau procédé permet de produire des protéines fixées au support immobilisées et des supports-protéines conjugués, tout en conservant au moins 50 % de l'activité biologique de la protéine ou du peptide. A cet effet, l'invention est caractérisée en ce qu'on fixe sur un support une protéine ou un peptide sous l'action de la transglutaminase en mettant en contact le support avec la protéine ou le peptide à fixer et avec la transglutaminase, et en ce que le support et la protéine agissent comme donneurs acyle et/ou amine et sont appropriés comme substrat de transglutaminase.
EP98945120A 1997-07-30 1998-07-30 Procede pour la fixation, catalysee par la transglutaminase, d'une proteine ou d'un peptide sur un support Withdrawn EP1001992A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE19732917 1997-07-30
DE1997132917 DE19732917C1 (de) 1997-07-30 1997-07-30 Verfahren zum Transglutaminase-katalysierten Koppeln von Protein oder Peptid an einen Träger
PCT/EP1998/004768 WO1999006446A2 (fr) 1997-07-30 1998-07-30 Procede pour la fixation, catalysee par la transglutaminase, d'une proteine ou d'un peptide sur un support

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EP1001992A2 true EP1001992A2 (fr) 2000-05-24

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WO2000058450A1 (fr) 1999-03-31 2000-10-05 Norbert Hampp Couplage covalent sans lieur de bacteriorhodopsine sous forme de membrane pourpre
JP3996307B2 (ja) * 1999-11-05 2007-10-24 富士フイルム株式会社 Ｄｎａ断片の固定方法、ｄｎａチップおよび核酸断片の検出方法
WO2007103288A2 (fr) * 2006-03-02 2007-09-13 Seattle Genetics, Inc. Conjugués anticorps-médicament modifiés
DE102006033167A1 (de) 2006-07-10 2008-01-24 Gelita Ag Verwendung von Gelatine und einem Vernetzungsmittel zur Herstellung eines vernetzenden medizinischen Klebers
DE102006033168A1 (de) 2006-07-10 2008-01-17 Gelita Ag Verwendung von Gelatine und einem Vernetzungsmittel zur Herstellung einer vernetzenden therapeutischen Zusammensetzung
SE540694C2 (en) * 2016-12-29 2018-10-09 The Univ Of Stavanger USE OF A TRANSGLUTAMINASE IN A METHOD FOR ENRICHING OR SCREENING FOR ONE OR MORE TARGET MOLECULES FROM A PRIMARY SOURCE

Citations (2)

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Publication number Priority date Publication date Assignee Title
EP0726317A2 (fr) * 1995-02-09 1996-08-14 Ajinomoto Co., Inc. Transglutaminase obtenu de Bacillus
EP0785276A1 (fr) * 1994-09-29 1997-07-23 Ajinomoto Co., Inc. Modification d'un peptide et d'une proteine

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WO1999006446A3 (fr) 1999-04-08
DE19732917C1 (de) 1998-10-15

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