EP0143807A1 - Method for immobilizing proteins or enzymes - Google Patents

Method for immobilizing proteins or enzymes

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Publication number
EP0143807A1
EP0143807A1 EP19840901761 EP84901761A EP0143807A1 EP 0143807 A1 EP0143807 A1 EP 0143807A1 EP 19840901761 EP19840901761 EP 19840901761 EP 84901761 A EP84901761 A EP 84901761A EP 0143807 A1 EP0143807 A1 EP 0143807A1
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Prior art keywords
proteins
protein
gene
enzymes
cloned
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EP19840901761
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German (de)
French (fr)
Inventor
Urs HÄNGGI
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Battelle Institut eV
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Battelle Institut eV
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Publication of EP0143807A1 publication Critical patent/EP0143807A1/en
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    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2468—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on beta-galactose-glycoside bonds, e.g. carrageenases (3.2.1.83; 3.2.1.157); beta-agarase (3.2.1.81)
    • C12N9/2471—Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01023—Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the invention relates to a method for immobilizing proteins, in particular enzymes, in which proteins are linked to carrier materials.
  • the object of the invention is therefore to provide a method which achieves the stability of the covalent linkage without the proteins or enzymes being inactivated thereby.
  • a product should be obtained in which the linkage can be made reversible.
  • the distribution of the reactive groups within a protein molecule is determined by the amino acid sequence.
  • the order of the amino acids is genetically fixed in the genetic mass of the genes and can be changed within a narrow framework using genetic methods. However, these methods do not allow a directed change, so that a clear redistribution of the amino acids responsible for fixing the proteins or enzymes can only be expected with great effort.
  • the known genetic engineering methods not only allow genes to be isolated and transferred to other organisms, but also to specifically modify gene elements, such as deleting parts of the information, for example Supplement information with other elements or substitute entire areas. With these interventions, the gene product, ie the protein or enzyme, can be specifically and specifically changed.
  • the genetic engineering methods are used to change the amino acid sequences of protein molecules in such a way that the distribution of the functional groups which can be used to link proteins to surfaces or support materials is designed in such a way that the biological activity of the proteins is achieved by the immbolization reaction if possible not lost.
  • cloned genes are cut using genetic engineering methods at one or more locations that are in nonessential areas of the proteins. Blocks of preferably n ⁇ 3 nucleotide pairs which code for a certain amino acid or amino acid sequence are inserted into these sites, where n is an integer.
  • the nucleotide blocks can e.g. code for lysine, thyrosine, acidic or other amino acids with functional side chains.
  • n x TGT / ACA or n x TGC / ACG blocks are installed. These nucleotides code for cysteine. In addition, there are the edge nucleotides, which ensure that the reading frame of the gene is not changed.
  • the insertion of the nucleotide sequences into the genes has the consequence that, when the genes are expressed, blocks of amino acids with functional side chains are incorporated into nonessentials of the proteins.
  • the ratio of the reactive groups inside and outside the activity centers of the proteins and the enzymes thus changes in such a way that greatly reduced or no inactivation rates are to be expected during the covalent immobilization.
  • the incorporation of cysteine blocks into the proteins also has the advantage that the immobilization takes place via disulfite bridges, which can be solved again simply by washing with mercaptans or other reducing agents. This ensures that protein or enzyme compounds with surfaces or carriers are formed which are at the same time stable against washing out, without losing their regenerability.
  • All materials which can be modified on the surface by methods known per se or which have reactive groups for covalent bonding from the start can be used as supports.
  • the reversible linkage is preferably carried out by oxidation / reduction reactions.
  • ⁇ -galactosidase The production of a fused gene is explained below using the example of ⁇ -galactosidase.
  • the gene of a ⁇ -galactosidase mutant is cloned in DNA vectors and cleaved with EcoRI.
  • a polynucleotide duplex with 6 ⁇ TGT / ACA, AATTGT / CA at the 5 'end and / TTAA at the 3' end is cloned into this cleavage site.
  • the gene modified in this way is then cloned into Escherichia coli bacteria, the product isolated and linked to a support, the surface of which has been modified to introduce SH groups, by oxidation.
  • the resulting disulfide bridges can easily be split again by reduction.
  • This example is shown schematically as follows.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Afin de pocéder à l'immobilisation de protéines, ou insère dans les parties non-essentielles de séquences d'acides aminés de molécules de protéines selon des techniques de génie génétique des blocs d'acides aminés avec des chaînes latérales fonctionnelles. On effectue ensuite la réticulation des parties insérées avec le matériau porteur.In order to pocede the immobilization of proteins, or inserts into non-essential parts of amino acid sequences of protein molecules according to genetic engineering techniques blocks of amino acids with functional side chains. The inserted parts are then crosslinked with the carrier material.

Description

================================================== ==================================================
Verfahren zur Immobilisierung von Proteinen oder Enzymen =================================================Process for immobilizing proteins or enzymes =========================================== ======
Die Erfindung betrifft ein Verfahren zur Immobilisierung von Proteinen, insbesondere von Enzymen, bei dem Proteine mit Trägermaterialien verknüpft werden.The invention relates to a method for immobilizing proteins, in particular enzymes, in which proteins are linked to carrier materials.
In weiten Bereichen der chemischen und medizininischen Analytik, der Medizintechnik und der industriellen Produktion von Feinchemikalien ist es wünschenswert, Proteine und Enzyme stabil mit Trägermaterialien zu verknüpfen. So ließe sich z.B. durch fixierte Enzyme, immobilisierte Antikörper oder gebundene Antigene der apparative Aufwand für analytische Tests verkleinern; Oberflächen, die mit physiologischen Systemen in Berührung kommen, könnten durch Proteinbedeckung körperverträglich gemacht werden; durch fixierte Enzyme könnten die Produktionsprozesse kontinuierlich gestaltet werden, was die Kosten für den Katalysator senken und die Abtrennung der Enzyme aus den Produkten überflüssig machen würde. Eine Vielzahl von Immobilisierungsmethoden ist bekannt. Diese bestehen in der physikalischen Adsorption von Proteinen an inerte TrägerSubstanzen, im Einschluß in die Poren von polymeren Gelen, in der Quervernetzung von Proteinen mit bifunktionellen Reagenzien und in der kovalenten Verknüpfung mit einem reaktiven, unlöslichen Träger.In broad areas of chemical and medical analysis, medical technology and the industrial production of fine chemicals, it is desirable to stably link proteins and enzymes with carrier materials. For example, fixed enzymes, immobilized antibodies or bound antigens could reduce the equipment required for analytical tests; Surfaces that come into contact with physiological systems could be made compatible with the body by protein covering; With fixed enzymes, the production processes could be continuously designed, which would lower the costs for the catalyst and make the separation of the enzymes from the products unnecessary. A variety of immobilization methods are known. These consist in the physical adsorption of proteins on inert carrier substances, in the inclusion in the pores of polymeric gels, in the cross-linking of proteins with bifunctional reagents and in the covalent linkage with a reactive, insoluble carrier.
Die Verwendbarkeit von proteinbedeckten Materialien und die Standzeiten enzymfixierter Reaktoren hängt wesentlich von der Stabilität der Protein-Trägerverbindung, derThe usability of protein-covered materials and the lifespan of enzyme-fixed reactors depends essentially on the stability of the protein-carrier compound
Resistenz gegen das Auswaschen und der Regenerierbarkeit des Systems ab. Keine der bekannten Immobilisierungsmethoden liefert ein Produkt, daß allen Anforderungen genügt. Die Gefahr des Auswaschens der Proteine bzw. Enzyme ist gering bei der kovalenten Verknüpfung der Proteine an Träger oder mit sich selbst. Die Verknüpfungsreaktionen beruhen alle darauf, daß die Proteine oder Enzyme über Amino-, Carboxy- oder andere funktionelle Gruppen der Aminosäureseitenketten in rein statistischer Weise mit den Verknupfungsoberflachen oder Trägern reagieren. Dabei läßt sich nicht vermeiden, daß Gruppen, die für die biologische Aktivität verantwortlich sind, ebenfalls reagieren. Damit geht die biologische Aktivität verloren. Die statistische Wahrscheinlichkeit, das biologisch essentielle Gruppe an der Verknüpfungsreaktion beteiligt sind, ist um so größer, je größer die Anzahl der funktionellen Gruppen innerhalb der Aktivitätszentren verglichen mit der Anzahl der Gruppen außerhalb der Zentren der Proteine und Enzyme liegen. Zudem ist das Regenerieren, d.h. der Austausch inaktivierter Proteinmoleküle durch neue, aktive Moleküle praktisch unmöglich. Die Regenerierung bereitet bei adsorptiven Prozessen in der Regel keine Schwierigkeiten. Die Stabilität der Protein-Trägerinteraktion ist jedoch gering. Bei den Einpolymerisierungs verfahren ist die Regenerierung nur in seltenen Fällen möglich. Zudem ist die Auswaschgefahr relativ groß.Resistance to washing out and the regenerability of the system. None of the known immobilization methods delivers a product that meets all requirements. The risk of washing out the proteins or enzymes is low with the covalent linkage of the proteins to the carrier or to itself. The linkage reactions are all based on the fact that the proteins or enzymes via amino, carboxy or other functional groups of the amino acid side chains in a purely statistical manner React way with the linking surfaces or vehicles. It cannot be avoided that groups which are responsible for the biological activity also react. The biological activity is lost. The greater the number of functional groups within the activity centers compared to the number of groups outside the centers of the proteins and enzymes, the greater the statistical probability that biologically essential groups are involved in the linking reaction. In addition, regeneration, ie the replacement of inactivated protein molecules with new, active molecules, is practically impossible. Regeneration is generally not a problem in adsorptive processes. However, the stability of the protein-carrier interaction is poor. When polymerizing regeneration is only possible in rare cases. In addition, the risk of washing out is relatively high.
Der Erfindung liegt daher die Aufgabe zugrunde, ein Verfahren bereitzustellen, das die Stabilität der kovalenten Verknüpfung erreicht, ohne daß dadurch die Proteine oder Enzyme inaktiviert werden. Es sollte ein Produkt erhalten werden, in welchem die Verknüpfung reversible gestaltet werden kann.The object of the invention is therefore to provide a method which achieves the stability of the covalent linkage without the proteins or enzymes being inactivated thereby. A product should be obtained in which the linkage can be made reversible.
Diese Aufgabe ist erfindungsgemäß dadurch gelöst, daß in die niσhtessentiellen Teile der Aminosäurensequenzen von Proteinmolekülen auf gentechnischem Wege Blöcke von Aminosäuren mit funktioneilen Seitenketten eingebaut und anschließend die eingebauten Teile mit dem Trägermaterial verknüpft werden. Vorteilhafte Ausführungsformen des Verfahrens sind in den Unteransprüchen 2 bis 5 beschrieben.This object is achieved according to the invention in that the non-essential parts of the amino acid sequences of protein molecules are genetically engineered to incorporate blocks of amino acids with functional side chains and then the built-in parts are linked to the carrier material. Advantageous embodiments of the method are described in subclaims 2 to 5.
Die Verteilung der reaktiven Gruppen innerhalb eines Proteinmoleküls ist durch die Aminosäuresequenz bestimmt.The distribution of the reactive groups within a protein molecule is determined by the amino acid sequence.
Die Reihenfolge der Aminosäuren ist genetisch in der Erbmasse der Gene fixiert und kann mit genetischen Methoden in einem engen Rahmen verändert werden. Diese Methoden lassen jedoch eine gerichtete Veränderung nicht zu, so daß eine deutliche Umverteilung der für die Fixierung der Proteine oder Enzyme verantwortlichen Aminosäuren nur mit aller größtem Aufwand zu erwarten ist.The order of the amino acids is genetically fixed in the genetic mass of the genes and can be changed within a narrow framework using genetic methods. However, these methods do not allow a directed change, so that a clear redistribution of the amino acids responsible for fixing the proteins or enzymes can only be expected with great effort.
Die bekannten gentechnischen Methoden erlauben es nicht nur, Gene zu isolieren und in andere Organismen zu übertragen, sondern auch Gen-Elemente spezifisch zu verändern, wie z.B. Teile der Information zu deletieren, die information mit anderen Elementen zu ergänzen oder ganze Bereiche zu substituieren. Mit diesen Eingriffen kann das Genprodukt, d.h. das Protein oder Enzym, spezifisch und gerichtet verändert werden.The known genetic engineering methods not only allow genes to be isolated and transferred to other organisms, but also to specifically modify gene elements, such as deleting parts of the information, for example Supplement information with other elements or substitute entire areas. With these interventions, the gene product, ie the protein or enzyme, can be specifically and specifically changed.
Erfindungsgemäß werden die gentechnischen Methoden dazu benutzt, die Aminosäuresequenzen von Proteinmolekülen so zu verändern, daß die Verteilung der funktioneilen Gruppen, die für die Verknüpfung von Proteinen an Oberflächen oder Trägermaterialien herangezogen werden können, derart gestaltet wird, daß durch die Immbolisierungsreaktion die biologische Aktivität der Proteine möglichst nicht verloren geht. Dazu werden klonierte Gene mit gentechnischen Methoden an einer oder mehreren Stellen geschnitten, die in nichtessentiellen Bereichen der Proteine liegen. In diese Stellen werden Blöcke von vorzugsweise n x 3 Nukleotidpaaren eingebaut, die für eine bestimmte Aminosäure oder Aminosäurefolge kodieren, wobei n eine ganze Zahl ist. Die Nukleotid-Blöcke können z.B. für Lysin, Thyrosin, saure oder andere Aminosäuren mit funktioneilen Seitenketten kodieren. Vorzugsweise werden n x TGT/ACA oder n x TGC/ACG-Blöcke eingebaut. Diese Nukleotide kodieren für Cystein. Hinzu kommen die Randnukleotide, die sicherstellen, daß das Leseraster des Gens nicht verändert wird.According to the invention, the genetic engineering methods are used to change the amino acid sequences of protein molecules in such a way that the distribution of the functional groups which can be used to link proteins to surfaces or support materials is designed in such a way that the biological activity of the proteins is achieved by the immbolization reaction if possible not lost. For this purpose, cloned genes are cut using genetic engineering methods at one or more locations that are in nonessential areas of the proteins. Blocks of preferably n × 3 nucleotide pairs which code for a certain amino acid or amino acid sequence are inserted into these sites, where n is an integer. The nucleotide blocks can e.g. code for lysine, thyrosine, acidic or other amino acids with functional side chains. Preferably, n x TGT / ACA or n x TGC / ACG blocks are installed. These nucleotides code for cysteine. In addition, there are the edge nucleotides, which ensure that the reading frame of the gene is not changed.
Der Einschub der Nukleotidsequenzen in die Gene hat zur Folge, daß bei der Expression der Gene Blöcke von Aminosäuren mit funktionellen Seitenketten in nichtessentielle der Proteine eingebaut werden. Damit verändert sich das Verhältnis der reaktiven Gruppen innerhalb und außerhalb der Aktivitätszentren der Proteinen und der Enzyme derart, daß bei der kovalenten Immobilisierung stark erniedrigte oder keine Inaktivierungsraten zu erwarten sind. Der Einbau von Cysteinblöcken in die Proteine hat überdies den Vorteil, daß die Immobilisierung über Disulfitbrücken erfolgt, die durch einfaches Waschen mit Merkaptanen oder anderen Reduktionsmitteln wieder gelöst werden können. Damit wird erreicht, daß Protein- bzw. Enzymverbindungen mit Oberflächen oder Träger entstehen, die gleichzeitig stabil gegen das Auswaschen sind, ohne daß die Regenierbarkeit verloren geht.The insertion of the nucleotide sequences into the genes has the consequence that, when the genes are expressed, blocks of amino acids with functional side chains are incorporated into nonessentials of the proteins. The ratio of the reactive groups inside and outside the activity centers of the proteins and the enzymes thus changes in such a way that greatly reduced or no inactivation rates are to be expected during the covalent immobilization. The incorporation of cysteine blocks into the proteins also has the advantage that the immobilization takes place via disulfite bridges, which can be solved again simply by washing with mercaptans or other reducing agents. This ensures that protein or enzyme compounds with surfaces or carriers are formed which are at the same time stable against washing out, without losing their regenerability.
Als Träger können alle Materialien eingesetzt werden, die durch an sich bekannte Methoden an der Oberfläche modifizierbar sind oder reaktive Gruppen zur kovalenten Bindung von Anfang an besitzen. Die reversible Verknüpfung erfolgt vorzugsweise durch Oxidation/Reduktionsreaktionen.All materials which can be modified on the surface by methods known per se or which have reactive groups for covalent bonding from the start can be used as supports. The reversible linkage is preferably carried out by oxidation / reduction reactions.
Die Herstellung eines fusionierten Gens wird im folgenden am Beispiel von ß-Galaktosidase erläutert. Hierzu wird das Gen einer ß-Galaktosidase-Mutante in DNS-Vektoren kloniert und mit EcoRI gespalten. In diese Spaltstelle wird ein Polynukleotid-Doppelstrang mit 6 x TGT/ACA, AATTGT/CA am 5'-Ende und /TTAA am 3'-Ende hineinkloniert. Das so veränderte Gen wird anschließend in Escherichia coli-Bakterien kloniert, das Produkt isoliert und mit einem Träger, dessen Oberfläche zur Einführung von SH-Gruppen modifiziert wurde, durch Oxidation verbunden. Die entstandenen Disulfid-Brücken können durch Reduktion wieder leicht gespalten werden. Dieses Beispiel wird schematisch wie folgt dargestellt. The production of a fused gene is explained below using the example of β-galactosidase. For this, the gene of a β-galactosidase mutant is cloned in DNA vectors and cleaved with EcoRI. A polynucleotide duplex with 6 × TGT / ACA, AATTGT / CA at the 5 'end and / TTAA at the 3' end is cloned into this cleavage site. The gene modified in this way is then cloned into Escherichia coli bacteria, the product isolated and linked to a support, the surface of which has been modified to introduce SH groups, by oxidation. The resulting disulfide bridges can easily be split again by reduction. This example is shown schematically as follows.
nicht essentiell für Aktivität not essential for activity
ThrMetlleThrAsnSerLeuAlaValValLeuGIn - ( N-Termi nus vo n ß-Gal) .. ..ATGACCATGATTACGAATTCACTGGCCGTCGTTTTACAA ... (5 ' -Ende des Gens ) . ..TACTGGTACTAATGCTTAAGTGACCGGCAGCAAAATGTT ...ThrMetlleThrAsnSerLeuAlaValValLeuGIn - (N-Termi nus vo n ß-Gal) .. ..ATGACCATGATTACGAATTCACTGGCCGTCGTTTTACAA ... (5 'end of the gene). ..TACTGGTACTAATGCTTAAGTGACCGGCAGCAAAATGTT ...
Spa l tung mit EcoR ISplitting with EcoR I
ATGACCATGATTACG AATTCACTGGCCGTCGTTTTACAA TACTGGTACTAATGCTTAA GTGACCGGCAGCAAAATGTTATGACCATGATTACG AATTCACTGGCCGTCGTTTTACAA TACTGGTACTAATGCTTAA GTGACCGGCAGCAAAATGTT
Insertion von ATTGTTGTTGTTGTTGTTGTTGTInsertion of ATTGTTGTTGTTGTTGTTGTTGT
CAACAACAACAACAACAACATTAA CAACAACAACAACAACAACATTAA
ATGACCATGATTACGAATTGTTGTTGTTGTTGTTGTTGTAATTCACTGGCCGTCGTTTTACAA TACTGGTACTAATGCTTAACAACAACAACAACAACAACATTAAGTGACCGGCAGCAAAATGTTATGACCATGATTACGAATTGTTGTTGTTGTTGTTGTTGTAATTCACTGGCCGTCGTTTTACAA TACTGGTACTAATGCTTAACAACAACAACAACAACAACATTAAGTGACCGGCAGCAAAATGTT
ThrMetlleThrAsnCysCysCysCysCysCysCysAsnSerLeuAlaValValLeuGln inserierter Bereich (Cys hat -SH) ThrMetlleThrAsnCysCysCysCysCysCysCysAsnSerLeuAlaValValLeuGln advertised area (Cys has -SH)

Claims

Patentansprüche Claims
1. Verfahren zur Immobilisierung von Proteinen, insbesondere von Enzymen, bei dem Proteine mit Trägermaterialien verknüpft werden, dadurch gekennzeichnet, daß in die nichtessentiellen Teile der Aminosäuresequenzen von Proteinmolekülen auf gentechnischem Wege Blöcke von Aminosäuren mit funktioneilen Seitenketten eingebaut und anschließend die eingebauten Teile mit dem Trägermaterial verknüpft werden.1. Process for the immobilization of proteins, in particular enzymes, in which proteins are linked to carrier materials, characterized in that in the nonessential parts of the amino acid sequences of protein molecules, blocks of amino acids with functional side chains are inserted by genetic engineering and then the built-in parts with the carrier material be linked.
2. Verfahren nach Anspruch 2, dadurch gekennzeichnet, daß das Gen des Proteins mindestens ein Mal in einem nichtessentiellen Bereich geöffnet und in diese Öffnung ein Block von Nukleotiden kloniert wird. 2. The method according to claim 2, characterized in that the gene of the protein is opened at least once in a non-essential area and a block of nucleotides is cloned into this opening.
3. Verfahren nach Anspruch 2, dadurch gekennzeichnet, daß n x 3 Basenpaare, vorzugsweise n x TGT/ACA oder n x TGC/ACG plus Randnukleotide zur Verhinderung einer Veränderung des Leserasters des Gens pro Öffnung kloniert werden, wobei n eine ganze Zahl bedeutet.3. The method according to claim 2, characterized in that n x 3 base pairs, preferably n x TGT / ACA or n x TGC / ACG plus edge nucleotides are cloned to prevent a change in the reading frame of the gene per opening, where n is an integer.
4. Verfahren nach Anspruch 2 oder 3, dadurch gekennzeichnet, daß das durch Hineinklonieren von Nukleotidpaaren veränderte Gen in einem Mikroorganismus kloniert, nach dem Züchten das veränderte Protein isoliert und auf chemischem Wege kovalent mit einem Trägermaterial verbunden wird.4. The method according to claim 2 or 3, characterized in that the gene modified by cloning in nucleotide pairs is cloned in a microorganism, after culturing the modified protein is isolated and chemically covalently linked to a carrier material.
5. Verfahren nach Anspruch 4, dadurch gekennzeichnet, daß das Trägermaterial durch Einfügung von SH-Gruppen an der Oberfläche modifiziert wird. 5. The method according to claim 4, characterized in that the carrier material is modified by the insertion of SH groups on the surface.
EP19840901761 1983-05-06 1984-04-26 Method for immobilizing proteins or enzymes Withdrawn EP0143807A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE19833316601 DE3316601A1 (en) 1983-05-06 1983-05-06 METHOD FOR IMMOBILIZING PROTEINS OR ENZYMES
DE3316601 1983-05-06

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JPS61124383A (en) * 1984-11-16 1986-06-12 Unitika Ltd Stabilization of immobilized fibrinolytic enzyme
US5958753A (en) * 1996-08-29 1999-09-28 The Wistar Institute Of Anatomy And Biology Nucleic acid sequences encoding Bau, a Bin1 interacting protein, and vectors and host cells containing same

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CA1200773A (en) * 1980-02-29 1986-02-18 William J. Rutter Expression linkers
BE882984A (en) * 1980-04-25 1980-08-18 Belge Etat BETA-GALACTOSIDASE IMMOBILIZATION PROCESS AND PRODUCTS THUS OBTAINED

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Title
See references of WO8404537A1 *

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