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WO1995003321A1 - Procede d'endomodification de proteins - Google Patents

Procede d'endomodification de proteins Download PDF

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Publication number
WO1995003321A1
WO1995003321A1 PCT/US1994/008127 US9408127W WO9503321A1 WO 1995003321 A1 WO1995003321 A1 WO 1995003321A1 US 9408127 W US9408127 W US 9408127W WO 9503321 A1 WO9503321 A1 WO 9503321A1
Authority
WO
WIPO (PCT)
Prior art keywords
protein
group
nucleophile
core
amino acid
Prior art date
Application number
PCT/US1994/008127
Other languages
English (en)
Inventor
Fred W. Wagner
Thomas R. Coolidge
Original Assignee
Bionebraska, Inc.
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 Bionebraska, Inc. filed Critical Bionebraska, Inc.
Priority to AU73382/94A priority Critical patent/AU7338294A/en
Publication of WO1995003321A1 publication Critical patent/WO1995003321A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/006—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length of peptides containing derivatised side chain amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
    • C07K1/1075—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of amino acids or peptide residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
    • C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/13—Labelling of peptides

Definitions

  • bioactive proteins can often be enhanced by their combination with other substances.
  • proteins can be immobilized to increase reaction efficiency and simplify the
  • proteins When used as detecting agents, proteins can be labeled to facilitate measurement. When used to complex with or treat biological organisms, proteins can be combined with bioactive agents (hereinafter called "augmentation") to help achieve treatment efficacy.
  • augmentation bioactive agents
  • Methods for immobilizing proteins are desirable because they localize reaction sites and improve
  • immobilized proteins are generally less susceptible to the loss of activity due to chemical attack and changes in temperature and pH than are free proteins.
  • Methods for labeling or augmenting proteins are desirable because they facilitate quantification, localization, specificity and reactivity of the protein.
  • the resulting combinations are the resulting combinations and/or powerful tools for clinical analysis and treatment.
  • Proteins can be physically adsorbed onto inert supports or can be covalently bound to the support through reaction with bifunctional linker arms.
  • Microencapsulation, gel entrapment and complexation (with ion exchange resins) also can bind and immobilize.
  • the present invention is an economical biochemical method for endomodification of proteins by immobilizing, labeling and augmenting them through endopeptidase transpeptidation reactions.
  • the method involves binding an immobilization support, label or bioactive agent (hereinafter called "auxiliary substance") to a protein core derived from a protein starting material composed of the core and a leaving group.
  • auxiliary substance an immobilization support, label or bioactive agent
  • the method of the invention involves two primary reactions. The first (hereinafter called the "primary transpeptidation reaction"),
  • nucleophile an amino acid derivative
  • primary binding reaction binds the side chain of the nucleophile to a specifically reactive group attached to the auxiliary substance.
  • the method of the invention can be practiced by alternative synthetic routes depending on whether the primary transpeptidation reaction or primary binding reaction is conducted first. These two routes are depicted in the scheme presented in the following section entitled Detailed Description of the Invention.
  • the first synthetic route can be employed with all sizes and solubilities of reactants.
  • a nucleophile having a distinctive side chain is first transpeptidated to the selected cleavage site of the protein by the primary transpeptidation reaction.
  • the transpeptidation involves contacting an endopeptidase enzyme, specific for an enzyme cleavage site, with a protein composed of at least one core linked by a cleavage site to a leaving unit, in the presence of a nucleophile.
  • endopeptidase enzyme cleaves the leaving unit from the core at the cleavage site and causes the core and the nucleophile to form an adduct of the core and
  • the resulting protein-nucleophile adduct is then bound to auxiliary substance by the primary binding reaction which takes advantage of the
  • the adduct either is directly bound to the auxiliary substance or is indirectly bound through a bifunctional linker arm. In either case, the binding reaction occurs between the side chain of the nucleophile and a specifically
  • the second synthetic route can be employed when the molarities of the reactants in the reaction medium are sufficient to permit relatively rapid enzymatic transpeptidation.
  • a nucleophile is first bound to the auxiliary substance by the primary binding reaction to form an intermediate of auxiliary substance and
  • nucleophile The binding can be accomplished by direct reaction of the side chain of the nucleophile and the auxiliary substance or indirectly through a linker arm that has been prebound to the auxiliary substance.
  • the intermediate is then transpeptidated to the protein core by the primary transpeptidation reaction described above. Transpeptidation with amino acid, peptide, polypeptide, or amino acid derivative nucleophiles can all be employed in this primary coupling reaction.
  • the protein starting material includes a core which may be a truncated version of its natural form.
  • the core may be truncated through deletion of amino acids at either, or both, of its C-terminal and N-terminal ends depending on the product desired.
  • the protein also includes a leaving unit linked to the core by an enzyme cleavage site recognized by the
  • the leaving unit may be one or more amino acid residues.
  • the protein starting material preferably is a biologically-active polypeptide. Included without limitation are enzymes, enzyme inhibitors, peptide hormones, DNA binding proteins, reading frame proteins, transcriptases, antibodies, F ab truncated antibodies, regulating proteins, peptides as small as two residues and various other functional proteins.
  • the protein starting material may as well be a fusion protein or a recombinant single copy polypeptide linked to a binding protein through an interconnecting peptide.
  • the protein starting material may also be a recombinant multicopy polypeptide such as multiple copies of the single copy polypeptide linked together with or without intraconnecting peptides. If an
  • intraconnecting peptide is present, it preferably has at least one site that is selectively cleavable by the endopeptidase cleavage enzyme.
  • the intraconnecting peptide may also serve as the leaving group from the C-terminal end of a single copy core polypeptide.
  • the binding protein aids in the purification of the
  • the preferred proteins for use in the inventive method are monoclonal or polyclonal antibodies.
  • the core constitutes the active fragments of such antibodies while the leaving group constitutes the constant region or some portion thereof.
  • antibodies include those that function to detect
  • antigens in biological systems or contaminants in biological or inanimate systems to carry bioactive agents to specific sites, to diagnose disease and organic disfunction, to separate antigens from other materials in biological or inanimate systems, and to remove antigens from biological or inanimate systems.
  • bioactive agents to carry bioactive agents to specific sites, to diagnose disease and organic disfunction, to separate antigens from other materials in biological or inanimate systems, and to remove antigens from biological or inanimate systems.
  • Especially preferred embodiments are mammalian
  • immunoglobulin proteins from the IgA, IgD, IgE, IgM, or IgG class of immunoproteins.
  • amino acid, peptide, polypeptide and amino acid derivative nucleophiles used in the method of the invention are respectively (1) an alpha amino acid having a side chain with a reactive substituent, (2) a short sequence of alpha amino acid residues with one of the residues having a side chain with reactive
  • N-terminal amino acid or amino acid sequence of the nucleophile is constructed or selected to be a reactive substrate for the endopeptidase.
  • nucleophile may have a simple, nonfunctional side chains in circumstances where they are also the auxiliary substance.
  • the side chain is chosen so that the nucleophile has a distinctive character relative to the amino acids of the protein.
  • the nucleophile rather than the amino acids of the protein is selectively and preferentially reacted with a specifically reactive group of the linker arm or auxiliary substance.
  • the second synthetic route such a
  • the linker arm used in the method of the invention is a flexible or semi-flexible chain which has as its termini (1) a specifically reactive group that is reactive with the side chain of the nucleophile and (2) an other functional group that reacts with a combining group of the auxiliary substance.
  • Immobilizing supports useful in the present invention are inorganic or organic materials which may be functionalized with a specifically reactive group for selective reaction with the side chain of the
  • the support is a porous or semiporous material that is biologically inert and insoluble in the medium used.
  • Bioactive agents include those that act to provide a desirable biochemical or therapeutic result. They may be functionalized with a specifically reactive group for reaction with the side chain of the
  • nucleophile or with a combining group that reacts with the other functional group of the linker arm.
  • chemotherapeutic agents include oxidizing or reducing agents, cytotoxic agents, anticancer agents, radioactive agents, antibiotics, antimicotics, anti-infectives, heavy metal agents, antiviral agents, lysing agents, chelating groups and the like.
  • Labels useful in the present invention include fluorescent groups, phosphorescent groups, colorimetric groups, radioactive groups, luminescent groups,
  • spectrometric groups nuclear magnetic resonance groups, electron spin resonance groups and other groups with physicochemical properties that may be detected by measuring means.
  • These labels may be functionalized with a specifically reactive group for reaction with the side chain of the nucleophile, or with a combining group that is reactive toward the other functional group of the linker arm.
  • the nucleophile may also function as a label when it carries radioactive atoms.
  • the endopeptidase enzymes used according to the method of the invention include those of the serine or cysteine peptidase class.
  • the endopeptidase enzymes trypsin and thrombin, of the serine peptidase class, and ficin and papin of the cysteine class, are especially desirable endopeptidase enzymes to serve as cleavage enzymes for the method of the invention.
  • amino acid residue cleavage site for the endopeptidase enzyme may be recognized by the amino acid residue cleavage site for the endopeptidase enzyme.
  • cleavage sites which are normally cleaved by an endopeptidase enzyme may be rendered less reactive or unrecognizable when adjacent to certain other amino acid residues. Use of this knowledge to render some cleavage sites less reactive is used advantageously to render substantial utility to endopeptidase enzymes which may otherwise be precluded from use in certain transpeptidation
  • the ability to cause combination of the nucleophile with the core is a desirable characteristic of the endopeptidase enzyme.
  • the entire transpeptidation process may be done in a single step under very mild conditions.
  • the number and sequence of steps of cleaving and reacting the starting material can vary depending on the starting material used.
  • nucleophile include control of pH, temperature,
  • the present invention is also directed to methods which employ the labeled, immobilized or
  • the method for use of the labeled protein involves combining of the labeled protein and the material upon which it is to act, removing any excess labeled protein and measuring the amount of labeled protein that has interacted with the material.
  • this method is useful for detection of antigens or enzymatic substrates/inhibitors by
  • the method for use of immobilized protein proceeds in a known manner as indicated by the character of the protein.
  • the protein preferably is an enzyme, antibody, DNA binding protein or regulatory protein.
  • the preferred uses will include enzymatically catalyzed reactions, antibody-antigen complexations, regulation of reactions and DNA or enzyme separations and/or
  • Another advantage is the ability to increase the packing density of the immobilized protein when all molecules are aligned in the same direction and have exposed active sites.
  • the method for use of a bioactive agent bound to a protein also proceeds in a recognized manner as indicated by the bioactive agent and the nature of the protein.
  • the action of the protein and bioactive agent cooperate to cause the effect desired.
  • the protein may act as a carrier to transport the agent across membranes or to cause its absorption into fluids, media or cells. It may also act as an absorption inhibitor to prevent transport of the agent across membranes or to prevent its absorption into fluids, media or cells. It may further act as a targeting vehicle to direct the agent to selective tissue sites or receptors.
  • auxiliary substance is bound. This control causes the auxiliary substance to bind to a specific site on the cleaved protein.
  • the present invention provides a process for the selective modification of a protein by
  • the present invention is based upon the discovery that amino acids, peptides, polypeptides, and amino acid derivatives nucleophiles can be transpeptidated into proteins, especially biologically active proteins by a
  • transpeptidation reaction under endopeptidase catalysis conditions (the primary transpeptidation reaction) .
  • the protein is composed of a core and a leaving unit such that during transpeptidation the nucleophile is
  • the auxiliary substance is bound to the protein core through either of two synthetic routes.
  • the nucleophile is separately transpeptidated to the protein core to form an adduct of the protein core and nucleophile.
  • the adduct is then bound to the auxiliary substance directly, or is bound indirectly through a linker arm-auxiliary substance combination.
  • nucleophile and auxiliary substance are directly bound, or
  • transpeptidation is defined as that process whereby a terminal amino acid or a chain of amino acid residues (leaving unit), linked through an endopeptidase enzyme cleavage site at the C-terminal end of a protein core, is replaced by another amino acid, peptide, polypeptide, or amino acid derivative (nucleophile), in the presence of an endopeptidase cleavage enzyme.
  • the method of the invention utilizes an endopeptidase enzyme, preferably of the serine or cysteine class, as the cleavage enzyme to catalyze the transpeptidation process.
  • the protein includes a core portion and a leaving unit.
  • the core is any useful polypeptide sequence such as a native sequence, a modified native sequence, a non-native sequence preferably having biological activity, transacted forms thereof and similar versions.
  • the leaving unit is linked to the core through an amide linkage and the amino acid residue or residues adjacent that linkage cause that linkage to be recognized as a cleavage site by the endopeptidase cleavage enzyme. According to the method of the
  • the core linked to a leaving unit may be derived from any source including natural sources, semisynthetic-natural sources, manipulation of natural sources, chemical synthesis, or protein expression.
  • the protein is contacted with at least one endopeptidase cleavage enzyme specific for at least one cleavage site.
  • the enzymatic cleavage of the protein at the linkage of the core portion and the leaving unit is conducted in the presence of a nucleophile.
  • sequence and number of steps in the transpeptidation can be varied depending upon the desired modification of the protein, the amino acid sequence of the desired product peptide, and the
  • the transpeptidation calls for the protein to be contacted with an endopeptidase cleavage enzyme, which has specific cleavage activity at the linkage between the core and the leaving unit.
  • the endopeptidase cleavage enzyme cleaves the leaving unit from the carboxy terminal of the core of the protein. Although it is not intended to be a limitation of the invention, it is believed that during this cleavage, the enzyme forms an acyl-enzyme
  • the enzyme causes the nucleophile to add to the cleaved core.
  • the nucleophile displaces the cleavage enzyme from the acyl-enzyme intermediate and links to the protein core where the leaving unit was linked.
  • the production of the modified protein is monitored by HPLC or other analytic procedure and the reaction is stopped by the addition of an acidic solution when the reaction has reached completion.
  • the enzyme cleavage site recognition sequence is not a sequence duplicated in the core or is not at an enzyme accessible sequence within the core.
  • the invention is further directed to modified enzyme cleavage sites which, when adjacent to certain amino acid residues, render the site unrecognizable or less reactive to cleavage.
  • modified enzyme cleavage sites which, when adjacent to certain amino acid residues, render the site unrecognizable or less reactive to cleavage.
  • the leaving units to be cleaved from the core are specifically chosen to provide a suitable leaving unit for the specific endopeptidase cleavage enzyme.
  • the nucleophiles are chosen to provide the amino acid or peptide chain to complete formation of the desired modified protein.
  • the first step is the primary transpeptidation reaction to form the adduct of protein core and nucleophile.
  • the choice of the particular nucleophile in the first step depends upon the identity of the amino acids of the protein and upon the distinctive character of the side chain of the nucleophile.
  • the side chain of the transpeptidated nucleophile acts as the binding site for the specifically reactive group of the attached
  • linker arm-attached substance or the combination of linker arm-attached substance. It has a structure that either is non-duplicative of the amino acids of the protein or is more highly reactive toward the specifically reactive group of the combination or auxiliary substance than are the amino acid side chains of the protein. It also is selected to avoid or minimize direct reaction with these side chains.
  • This selectivity imposed by the nucleophile side chain is accomplished by its reactive substituent.
  • This substituent may be a sulfhydryl, olefinyl, amino, azidyl, hydrazinyl, epoxy, hydroxyl, activated hydroxy wherein the activator is a facile leaving group such as tosyl, mesyl and benzoyl, an acid group such as
  • amidinyl imidazo, pivaloyl ester, neopentyl ester and the like, phosphoramidoyl, ferrocenyl, ferro complexes, boronyl and similar reactive functional groups.
  • the primary binding reaction is accomplished by binding the
  • nucleophile side chain either to the auxiliary substance or to its combination with the linker arm.
  • specifically reactive group of the combination or auxiliary substance correlates with the reactive substituent of the side chain so that the side chain and auxiliary substance or combination readily react without substantially involving other groups of the protein.
  • the reactive substituent and specifically reactive group are correlated as pairs of groups.
  • Several embodiments of this pair exhibit non-competitive binding which essentially will not involve other groups of the protein. These include, for
  • an organomercuric group or Alman reagent which are particularly useful with antibodies because antibodies do not naturally contain free sulfhydryl groups, i.e. cysteine within or close to their active sites;
  • reaction medium e.g. a teflon-coated iron wire coil, which form a magnetic transpeptidate
  • a chelating group and a chelated moiety such as ethylene diamine tetraacetate and a transition metal, which form a chelate
  • a polar olefinic or substituted olefinic group and the corresponding monomer which polymerize by free-radical means under mild conditions, e.g. N-acryloyl lysine and acrylamide, and form a polymer;
  • radical stabilizing group having a radical- labile C-H bond such as benzoylphenylalanine and a benzyl, allyl, or arylalkyl group, which can be photolyzed to form an adduct between the keto carbon and the C-H carbon of the free radical stabilizing group.
  • free radical stabilizing groups as polyamide, polycinnamide, polystyrene, or fluorene
  • substantially selective reaction of the side chain and attached substance or combination include, for example:
  • an aromatic amino group and an epoxy, activated ester or aldehyde group preferably an aromatic epoxy or aldehyde group, which can be reacted to form a nitrogen-carbon adduct, and under slightly acidic conditions to protonate the amine groups of the protein;
  • concentrations of reactants The procedures will generally involve stirred reactors, removal of side products and slow addition of reagents.
  • a further condition is the maintenance of a minimal concentration of any reactant that can react with more than one group in the reaction mixture. For example, a minimum
  • the nucleophile and the auxiliary substance or its combination with linker arm are first bound by the primary binding reaction to form an intermediate.
  • This step can be accomplished by employing (1) any of the reactive substituent and specifically reactive group pairs described above; (2) the combining group and other functional group pairs described below for the linker arm, or (3) by any of the known methods for forming an amide, ester, ether, imino, carbonate, urethane (carbamate), carbon-carbon, carbonnitrogen, sulfur-carbon, sulfur-oxygen-carbon or carbonoxygen bond. Methods to form these bonds and the particular groups formed thereby are known in the art. See, for example "Chemical Reagents for Protein
  • nucleophile and auxiliary substance or linker arm combination will suffice because this binding reaction is not conducted in the presence of the protein.
  • the second step of route 2 transpeptidates the intermediate to the protein core through the primary transpeptidation reaction of the nucleophile portion of the intermediate and the protein (core and leaving unit). It is accomplished by endopeptidase catalysis under hydrolytic avoiding conditions. The selectivity of this reaction suits it as the one to be conducted in the presence of protein.
  • the scheme of route 2 utilizes this feature to best advantage because it places the primary transpeptidation reaction last in the reaction sequence thereby eliminating the potential interference from the primary binding reaction.
  • the second step of route 2 has some attendant parameters that primarily are directed to reaction efficiency.
  • the reactants should have sufficient solubility in the reaction medium to enable relatively facile transpeptidation to take place. Generally, this solubility will be preferably about 0.05 to 2M for the reactants and 1 to 100 ⁇ M for the enzyme. When the solubilities of the reactants of the transpeptidation reaction are less than this, route 1 is preferentially employed.
  • the linker arm version of routes 1 and 2 would be selected according to the invention.
  • the environment of the protein may prevent approach of a large, bulky auxiliary
  • the auxiliary substance may not contain functional groups that are specifically reactive with the side chain of the nucleophile.
  • the linker arm increases the distance between the auxiliary substance and the protein which can help maintain the activity of the protein.
  • the protein can more freely adopt a spatial conformation that is appropriate for its reactivity. Fifth, it will lessen or minimize alteration of protein confirmation caused by auxiliary substance proximity.
  • the structure of the linker arm includes functional groups at the ends of a flexible to semi-flexible chain.
  • One of the functional groups is the specifically reactive group mentioned above that reacts with the side chain of the nucleophile.
  • the other functional group of the linker arm is chosen to readily react with available combining groups on the auxiliary substance. Of course, this pair of groups of the linker arm is selected so that one does not substantially interfere with the other when they are used in the binding and combining reactions.
  • the step to combine linker arm and attached substance is accomplished before the binding reaction with the nucleophile. Since protein and nucleophile are not present during this step, the kinds of reactions available are numerous. If the combining group of the auxiliary substance is an aldehyde group, the other functional group may be an amine (Schiff base product), an activated acid such as an iminocarboxy, carboxyalkoxy or acid halide (amide product) or epoxy (substituted amine product).
  • the other functional group may be an activated acid or ester (ester product) or activated alkyl such as a halo alkyl, alkyl tosyl or alkyl mesyl (ester
  • the combining group of the auxiliary substance is an acid group
  • the other functional group may be an amine (amide product) or activated hydroxyl (ester product). If the combining group of the
  • auxiliary substance is a chelating agent
  • the other functional group may be a bound metal group.
  • Other pairs of reactants include water soluble carbodiimide and amino; N-acyl succinimide and amino; and olefin and diene as well as those described above under part (3) of the primary binding reaction for route 2. Of course, the reverse order of reaction is also possible.
  • the backbone of the linker arm may be any that provides a flexible or semi-flexible chain. Included are polymers and oligomers of amides (peptides),
  • the length of the backbone may be from about two to about 100 atoms or monomeric units, preferably about four to about 20 atoms or monomeric units in length.
  • Examples of the backbone include hexylenyl, decylenyl, poly(4-aminobutyric acid)
  • proteins can be transpeptidated according to the present invention.
  • suitable proteins include enzymes, enzyme inhibitors, hormones including peptide hormones, antibodies, F ab truncated antibodies, functional proteins, transcriptases, reading frame proteins, DNA binding proteins, and other biologically active polypeptides.
  • monoclonal or polyclonal antibodies, DNA binding proteins, enzymes, and reading frame proteins are preferred as proteins. They are generally useful in the diagnosis of diseases, disorders, or hereditary dysfunctions.
  • the antibodies are also generally useful in separation techniques and for detection of antigenic material. This includes mammalian immunoglobulin proteins from the IgA, IgD, IgE, IgM, or IgG class of immunoproteins.
  • mono or polyclonal antibodies and enzymes are preferred as proteins .
  • histocompatibility proteins polypeptide inhibitors, peptide toxins, structural proteins (e.g. collagen), globular proteins and fibrous proteins are preferred as proteins.
  • nucleophiles can be used in the present invention. Generally these include alpha amino acids or amino acid sequences (peptides,
  • polypeptides with neutral, basic, or acidic side chains wherein the side chains may contain the reactive
  • immobilizing support, or binding bioactive agents to proteins is generally coordinated with the enzyme and the protein chosen.
  • trypsin will generally transpeptidate nonacidic amino acids onto Arg or Lys residues of a protein.
  • Certain other enzymes which utilize cysteine and/or serine at their enzymatic sites and are derived from plant and microbial sources, will transpeptidate amino acids with acidic side chains to proteins.
  • the amino acid nucleophile will exhibit a distinctive reaction character. This will allow its selective binding to the auxiliary substance or its combination with linker arm. The distinctive character results from the reactive substituent of the side chain of the nucleophile as explained above.
  • This reactive substituent may be a sulfhydryl, hydroxy, activated hydroxyl, phosphoramidoyl, hydrazinyl, amino, azidyl, epoxy, acid, boronyl, activated esters, ferrocentyl, ferro complex, or olefinyl group, mixtures thereof and other functionally reactive groups.
  • monocarboxylic acids e.g., glycine, alanine, valine, norvaline, leucine, isoleucine, and norleucine (useful as radioactive label); hydroxy amino acids such as serine, threonine, and homoserine; sulfur-containing amino acids such as methionine, cystine, cysteine, and taurine (for linker arm or auxiliary substance binding); diamino monocarboxylic acids such as orthinine, lysine, and arginine (for linker arm or auxiliary substance binding); and monoamino dicarboxylic acids such as aspartic acid and glutamic acid (for linker arm or auxiliary substance binding).
  • aromatic amino acids such as phenylalanine and tyrosine
  • heterocyclic amino acids such as histidine and tryptophan
  • olefinic amino acids such as 2-amino-2-vinyl acetic acid (for linker arm or auxiliary substance binding) are included within the group of amino acids of the present invention.
  • Additional amino acids are those with the C-terminal end protected. This includes, for example, amides, anilides, hydrazides, esters, and the like.
  • Embodiments of peptide nucleophiles include various sequences of the foregoing amino acid residues as well as any about two to about 50 unit sequence of the twenty naturally occurring amino sequence. Examples include Gly Arg Mea or Gly-Biotin where Mea is maleic acid.
  • nucleophile examples include aliphatic amino acids, hydroxy amino acids, their activated derivatives, phosphoramidoyl amino acids, sulfur-containing amino acids, diamino monocarboxylic acids, activated ester amino acids, aromatic amino acids, and heterocyclic amino acids.
  • preferred embodiments include serine, alanin,
  • Labels for the proteins according to the present invention include labeled, or tagged amino acids having a variety of substituents or atoms that possess properties suitable for detection by conventional techniques. Such properties include photoaffinity, magnetism, radioactivity, fluorescence, enzymatic activity, electron dense (x-ray), nuclear magnetic resonance, electron spin resonance, antigenicity, and phosphorescence.
  • amino acids can be labeled with either 14 C or 3 H atoms. Further, the amino acids may be tagged by known fluorescent dyes,
  • porphyrins colorimetric dyes, reactive groups and antigens or enzymatic substrates that permit
  • Immobilizing supports useful in the present invention are inorganic or organic materials
  • the support will be functionalized with the combining group for the other functional group mentioned above.
  • the reactive substituent may be chelating ferromagnetic groups.
  • the immobilizing support then has the appropriate character to produce binding.
  • the support may be magnetic wire that is rendered inert to the reaction medium, e.g. with teflon. Passing a current through the wire will establish the magnetism needed to cause binding.
  • a magnet external to the system i.e. outside the chromatographic medium
  • the support may be an immobilized metal or other
  • the support may be a porous or semiporous solid. Preferably, it is biologically inert and
  • Materials that may be used as supports include fibers, sheets, microspheres, particles, beads, membranes, and the like.
  • the surface of the immobilizing support of the present invention is preferably porous.
  • substantially spherical polymeric beads or microspheres of agarose allows large surface areas for the attachment of protein at high density.
  • a surface is considered porous where the size of the majority of the pores in the material is sufficiently large so as to allow the migration of the protein into the interior of the spheres.
  • the size and shape of the support may be varied widely, depending on the particular protein and its intended use.
  • the immobilizing supports include a wide variety of substances.
  • the choice of support depends upon the choice of the nucleophilic and/or linker arm as well as on the intended use of the
  • transpeptidation reactions nucleophile, specifically reactive group and reactive group all are compatible as described above.
  • the support is chosen such that the
  • nucleophile will readily transpeptidate to the support or support-linker arm combination in preference to any other reactive sites on the protein.
  • cysteine may be used as the amino acid nucleophile to transpeptidate with a protein with no sulfhydryl groups, e.g. an antibody.
  • a support or support-linker arm specifically reactive group is chosen that would react with the sulfhydryl moiety, for example, an
  • organometallic group such as an organo mercury compound.
  • 2-amino-hex-4-enoic acid may be the amino acid nucleophile, and a specifically reactive group for the support may be one that would specifically react with the unsaturated side chain, as for example through a Diels Alder reaction.
  • a photoaffinity label such as N-hydroxy succinimidyl-4-azidosalicylic acid side chain, and an arylamine as the specifically reactive group on the attached substance.
  • This salicylic side chain is to be transpeptidated to the epsilon amino group of a lysine before the photo addition so that it will not be
  • Photoreaction under, for example, u.v. light will accomplish the desired photo binding reaction.
  • linker arm available groups on the support act as the reactive group.
  • the other functional group of the linker arm is appropriately chosen to bind with the reactive group.
  • bioactive agent a bioactive agent to a protein at a site remote from the active site.
  • bioactive agents can be carried or transported by the protein to a site where they can perform a desired reaction.
  • the bioactive (biologically active) agent includes physiologically or pharmacologically active substances that act locally or systemically in the body.
  • biologically active agents include peptide drugs, protein drugs, desensitizing agents, antigens, vaccines, anti-infectives, antibiotics, antimicrobials, antiallergenics, steroidal anti-inflammatory agents, decongestants, miotics, anticholinergics,
  • estrogens progestational agents, humoral agents, prostaglandins, analgesics, antispasmodics,
  • antimalarials antihistamines, cardioactive agents, nonsteroidal anti-inflammatory agents, antiparkinsonian agents, antihypertensive agents, /3-adrenergic blocking agents, nutritional agents, metal compounds, anti-cancer compounds such as fluorinated nucleotides, nucleotide analogs, cytosine arabinocide, 5-fluorouracil, ricin-A, tetanus toxin, cyclic therapeutic peptides such as anamycin, erythromycin, cyclosporin, AZT, and alkaloids.
  • various forms of the biologically active agents may be used. Forms such as uncharged molecules,
  • bioactive agents are functionalized to carry specifically reactive groups for transpeptidation to the nucleophile directly.
  • appropriate available combining groups on the bioactive agent can be reacted with the other functional group on a linker arm.
  • this functionalization will be accomplished with a group already present within the agent.
  • the cleavage enzymes include the class of endopeptidases.
  • the endopeptidases suitable for use in the present invention include the serine and cysteine peptidases.
  • the mechanism of action of serine and cysteine endopeptidases is believed to involve the formation of an acyl-enzyme intermediate with the core after cleaving the leaving unit. Under appropriate reaction conditions, it is believed that the nucleophile acts as a nucleophile and displaces the endopeptidase cleavage enzyme from the acyl-enzyme intermediate.
  • Serine peptidases are in a group of animal and bacteria endopeptidases which have a catalytically active serine residue in their active center.
  • endopeptidases of the serine peptidase classification include trypsin and thrombin.
  • the endopeptidase trypsin is found in the pancreas of all vertebrates. It is released via the pancreatic duct into the duodenum as a trypsinogen.
  • Trypsin is known for its pronounced cleavage site specificity, catalyzing hydrolysis of only Lys-X and Arg-X (X is another amino acid residue) bonds.
  • Trypsin's affinity for cleavage at the Lys-X bond is significantly diminished when immediately adjacent to an amino acid containing a carboxylic acid side chain, specifically including the amino acids glutamic acid and aspartic acid.
  • a discovery of the present invention utilizes the knowledge of decreased cleavage activity at the Lys-X cleavage sites which are adjacent to an amino acid containing a carboxylic acid side chain. This discovery has rendered the endopeptidase trypsin of great utility in the formation of modified proteins, according to the method of the invention. Natural or synthetic forms of thrombin are suitable for the method of the invention.
  • glycoprotein endopeptidase thrombin also of the serine peptidase classification, is responsible for the conversion of fibrinogen to fibrin. It is naturally produced during blood coagulation by the action of factor X a upon prothrombin. This endopeptidase has considerable sequence homology with trypsin and contains the catalytically important residues His, Asp, and Ser in the B chain. Thrombin has a cleavage
  • Thrombin is known for its cleavage site specificity at the carboxyl side of the Arg- residue.
  • the known recognition sequence for the cleavage site Arg-X is Gly-Pro-Arg.
  • a discovery of the present invention is that thrombin also cleaves at the carboxyl side Arg- residue within the recognition sequence of Gly-Ala-Arg. This discovery enhances the use of
  • the transpeptidation process according to the method of the invention may be accomplished using starting protein derived from single or multicopy constructs, or single or multicopy fusion protein constructs.
  • the conditions for the primary transpeptidation reaction efficiently favor transpeptidation over peptide cleavage. As can be seen from the following discussion of the application of the primary transpeptidation reaction to antibody protein, these conditions generally involve control of Ph, temperature, reactant
  • the conditions for transpeptidation are within range of about pH 2 to 11.
  • transpeptidation occurs between 5 seconds and 1.4 hours.
  • the reaction temperature is the functional range of the enzyme, preferably up to about 40°C.
  • the concentrations of the reactants and enzyme are adjusted to provide optimum results. Generally, the highest possible concentrations of enzyme, nucleophile and protein are used that coincide with an appreciable primary transpeptidation reaction rate.
  • the protein is present at a concentration of from about 1 ⁇ M to about 1M, especially up to about 1 ⁇ M when the protein is an antibody.
  • the nucleophile or intermediate incorporating the nucleophile is preferably present at a concentration of at least 0.05 molar and especially a concentration of from about 0.1 to 2 molar.
  • the enzyme is preferably present at a concentration of about 1 to 100 ⁇ M, preferably about 1 to 100 ⁇ M.
  • the transpeptidation can also be facilitated by conducting the reaction in substantially all organic solvent and a minimum of water, e.g., 5-20% water.
  • Useful organic solvents include DMF, DMSO, dimethyl acetamide, 1,4-butanediol and the like.
  • the incubation time (reaction time) of the protein and the nucleophile is from about 0.2 to 10 hours, preferably from 1.0 to 8 hours for condensation while for transpeptidation or transesterification, it is from about 30 seconds to about 1.5 hours.
  • polypeptide however, it is understood that the method of the invention is suitable for transpeptidation of polypeptides, regardless of the source.
  • the transpeptidation process of the invention is preferably a one step reaction conducted in a buffer solution capable of maintaining pH at about pH 2-11, preferably pH 3-10, and more preferably pH 5-9.
  • Suitable buffers for the present invention include Mes, Tris, Hepes, Capso, and the like.
  • the modified recombinant polypeptide for example tagged Glucagon-like Peptide 1 (GLP1) (7-36)-HN C 6 H 3 I 2 128 2 , is produced.
  • the recombinant polypeptide GLP1 (7-34) core with Lys 3A linked to the leaving unit -Ala-Phe-Ala with the Lys 3A -Ala 35 as the cleavage site is dissolved in buffer.
  • transpeptidation mixture is added the suitable addition unit, containing desired amino acid or peptide sequence, in a concentration designed to provide at least a stoichiometric amount, preferably a 100 M excess in an amount relative to the recombinant polypeptides.
  • Gly-Arg-NH C 6 H 3 I 2 128 is desired sequence which is suitable addition units for synthesis of the modified recombinant polypeptide product GLP1 (7-36)-NHC 6 H 3 I 2 128 .
  • the cleavage enzyme trypsin is added to the reaction mixture in a trypsin:polypeptide molar ration of about 1:2000, and more preferably 1:1000.
  • the production of the modified recombinant polypeptide product GLP1 (7-36)-NHC 6 H 3 I 2 128 is monitored by HPLC or by capillary electrophoresis and the reaction stopped by the addition of an acid solution. Suitable acid solutions for stopping the reaction include HCl and the like.
  • the modified recombinant polypeptide product is purified from the mixture by HPLC, Ion exchange
  • the recombinant polypeptide product may be used immediately or may be stored at -20 tp -80.
  • thrombin Another example of endopeptidases which may act as a cleavage enzyme according to the method of the invention is thrombin.
  • thrombin has a cleavage site preference for -Arg-X.
  • the known recognition sequence for the -Arg-X cleavage site is Gly-Pro-Arg.
  • a discovery of the present invention is that thrombin also recognizes the cleavage recognition sequence Gly-Ala-Arg. The discovery of this recognition sequence renders the endopeptidase enzyme thrombin of greater utility in preparation of modified proteins by the method of this invention and other recombinant methodologies.
  • the transpeptidation process of the present invention utilizing the endopeptidase enzyme thrombin is a one pot reaction conducted in a buffer solution capable of maintaining pH at about pH 2-11, preferably pH 3-10, and more preferably pH 5-9. Suitable buffers for the present invention are as previously described for trypsin.
  • the protein includes a GRF (1-41) core linked to an -Ala-leaving unit.
  • a suitable nucleophile for synthesis of GRF (1-44)-Mea is Ala-Arg-Leu-Mea.
  • thrombin recognizes the cleavage site -Arg-X within a Gly-Ala-Arg
  • the protein GRF (1-41)-Ala is buffer combined with a suitable nucleophile in at least a stoichiometric ratio with the polypeptide.
  • thrombin is added to the mixture in a
  • thrombin:polypeptide molar ratio of about 1:5000, preferably 1:1000, and more preferably 1:3000.
  • GRF (1-44)-NH 2 The production of GRF (1-44)-NH 2 is monitored by
  • HPLC HPLC or other appropriate analytic technique and the reaction stopped by the addition of an acid solution.
  • Suitable acid solutions for stopping the reaction include Hcl, acetic acid and the like.
  • the modified protein is purified from the reaction mixture by
  • the protein product may be used immediately or may be stored as a
  • Anti-asparagine synthetase monoclonal antibodies and anti-Fl ATPase monoclonal and polyclonal antibodies can be obtained from laboratory stocks.
  • Monoclonal antibody stocks can be obtained in the form of mouse Ascites tumor fluids and polyclonal antibody stocks can be obtained from rabbit serum.
  • Antibodies from either source can be purified by addition of solid ammonium sulfate to a concentration of 50% of the saturation level.
  • the precipitated protein can be collected by centrifugation and dissolved in a minimal amount of 10 mM Tris-HCl [Tris,
  • the preparation can be subjected to a second ammonium sulfate treatment until a 50% saturation level is reached.
  • the precipitate can be collected by
  • the purified antibodies were dissolved in a minimal amount of water and dialyzed for 18 to 24 hours against 10 mM sodium bicarbonate at 4oC.
  • the purified antibodies can be stored in aliquots at -20oC until needed.
  • antibody as used herein means anti-asparaginine synthetase monoclonal antibody, and anti-F1 ATPase mono and polyclonal antibodies.
  • the peptide Gly-Asn-NHC 6 H 3 I 2 128 can then be prepared by the following technique.
  • the dipeptide Gly-Asn- can be synthesized by chemically protecting the amine group of Gly and coupling to Asn with carbodiimide in DMF.
  • the protected dipeptide can then be amidated with 3,5 diiodo aniline by suitable amidating conditions such as carbodiimide or thionyl chloride.
  • Radioactive peptides can be diluted with unlabeled amino acid to a specific activity of 0.5 to 2 mCi per millimole.
  • the diluted amino acid is then purified by repeated precipitation with ethanol at -20°C.
  • the specific activity of the diluted, purified amino acid can be determined as follows, and used in subsequent calculations of label incorporation.
  • a known volume of the amino acid solution can be diluted to 1.0 ml with 0.1 M sodium phosphate (pH 6.8). This diluted sample can be counted in a Beckman LS-100 liquid scintillation counter (Beckman Instruments, Fullerton, CA) with 10.0 ml 3a70B scintillation fluid (Research Products, Elkgrove, IL).
  • the amino acid concentration of the stock amino acid solution can be determined on a known volume by assay of the amino groups with the ninhydrin assay; see S. Moore et al., J. Biol. Chem., 157, 367 (1948).
  • a standard solution can be prepared for this assay by dissolving glycine in water at 0°C to obtain a saturated solution. The liquid is separated from any undissolved solid glycine, warmed to room temperature, and used as a standard. The concentration of the standard was assumed to be 1.89 M; see J. B.
  • Example 2 peptide stock solutions can be made and diluted with water to the desired concentrations. From this solution a 4 ⁇ l portion can be removed and diluted to 1.0 ml with 0.1 M sodium phosphate (pH 6.8). The concentration of the antibody in the solution can be calculated assuming a standard absorbance of 1.46 absorbance units per mg of antibody; see A. Good et al., in Selected Methods in Cellular Immunology, Mishell & Shiigi eds., W. H.
  • the concentration of the peptide in the incubation mixture can be determined by counting a small sample of the diluted mixture following further dilution to 1.0 ml with 0.1 M sodium phosphate (pH 6.8).
  • the undiluted mixture can be divided into equal parts.
  • To one of these portions can be added the endopeptidase papion and sufficient organic solvent (DMF or 1,4 butanediol to make a 90% solution).
  • the other portion serves as a control and can be diluted similarly with water. Once these two solutions are prepared, they can be incubated in a 37°C water bath for
  • the samples can be then removed from the water bath and either analyzed immediately or frozen and used as soon as possible.
  • the resulting product is F(ab) 2 labelled with the iododipeptide.
  • a GLP1 peptide is a 36 amino acid peptide that has been recombinantly produced but without a mechanism for providing for the amidation of the C-terminal arginine residue.
  • the method of the invention has been designed to produce a single copy fusion protein construct containing one copy of a gene encoding a truncated core GLP1 and biotinylating the core GLP1 by a transpeptidation reaction, using the endopeptidase trypsin, to form an immobilizable GLP1 polypeptide.
  • the strategy involves forming a DNA construct encoding a single copy recombinant fusion protein.
  • the single copy fusion protein includes at least three segments.
  • the first segment is a binding protein which exhibits strong reversible binding to a specific small molecular weight ligand.
  • the second segment is an interconnecting peptide which is selectively cleavable by an enzyme or chemical technique.
  • the third segment is a variable fused peptide containing one copy of the desired natural or synthetic polypeptide, in this case GLP1 ( 7-34 ) .
  • GLP1 7-34
  • the single copy fusion protein can be formed with human carbonic anhydrase modified at residue 240 as the binding protein.
  • the modification of carbonic anhydrase at residues 240 involves a substitution of a leucine for a methionine.
  • the interconnecting peptide is a methionine residue which can be cleaved by cyanogen bromide.
  • the variable fused polypeptide contains a single copy of a modified truncated GLP1 (7-37) peptide having Lys at 26, Glu at 27, Lys at 34 and Ala Phe Ala at 35, 36 and 37.
  • the core GLP1 peptide is truncated from the native sequence so that it contains residues
  • this single copy recombinant fusion protein can be produced from a DNA construct formed as follows.
  • the DNA sequence from the human carbonic anhydrase II gene is modified so that the methionine codon at amino acid residue 240 is replaced with a leucine codon using site directed mutagenesis as described by Sambrook et al., in Molecular Cloning, "A Laboratory Manual", Cold Spring Harbor, N.Y. (1989).
  • the modified gene for human carbonic anhydrase is then cloned into an expression vector which is compatible with E. coli.
  • a short DNA fragment including the codon for methionine is chemically synthesized and inserted immediately downstream from the end of the gene for human carbonic anhydrase by standard methods.
  • a DNA sequence encoding the truncated core GLP1 (7-34)-Ala-Phe-Ala polypeptide is formed by automated DNA synthesis and inserted directly downstream from the interconnecting DNA segment encoding the methionine codon.
  • the final recombinant expression vector encoding the single copy fusion protein is transformed into E. coli by standard methods and the expressed recombinant single copy fusion protein can be obtained using
  • Cleavage and transpeptidation can be conducted as follows. For example, a 40 mg/ml solution of HCA-Met-GLP1 (7-34)-Ala-Phe-Ala can be digested with a 50-fold excess of cyanogen bromide (CNBr) methionine in 70% formic acid to release the GLP1 (7-34)-Ala-Phe-Ala peptide.
  • the reaction mixture can be incubated in the dark under oxygen-free nitrogen at 20°-25°C for 16-24 hours.
  • the reaction mixture is diluted with 15 volumes of water and freeze dried. For the complete removal of acid and by-products, the freeze drying can be repeated after further addition of water.
  • This cleavage reaction yields human carbonic anhydrase and the recombinant GLP1 (7-34)-Ala-Phe-Ala polypeptide.
  • the cleaved GLP1 7-34 Ala-Phe-Ala polypeptide can be separated from human carbonic anhydrase by normal chromatographic methods, i.e., ion exchange, reverse phase, or by size exclusion.
  • the cleaved GLP1 (7-34)-Ala-Phe-Ala polypeptide can be separated from the human carbonic anhydrase by simple chromatographic methods, i.e., ion exchange, reverse phase, or by size exclusion.
  • the cleaved GLP1 (7-34)-Ala-Phe-Ala polypeptide can be separated from the human carbonic anhydrase by simple
  • methionine, and peptide is diluted with water to a protein concentration of 20 mg/ml while maintaining an acetic acid concentration of 10%.
  • the carbonic anhydrase can be quantitatively precipitated and greater than 80% of the peptide remains in solution.
  • the supernatant can be applied to an open C-8 column which is rinsed with four column volumes of 10% acetic acid.
  • the GLP1 (7-34)-Ala-Phe-Ala can be eluted from the column with 50% acetonitrile in 10% acetic acid.
  • the peptide can then be freeze dried.
  • the GLP1 (7-34)-Ala-Phe-Ala can be transpeptidated to yield the modified recombinant native GLP1 7-36-NH 2 amino acid product as follows.
  • the recombinant GLP1 (7-34)-Ala-Phe-Ala polypeptide can be cleaved with trypsin at the cleavage site between amino acid residues 34 and 35 at the Lys-Als bond in the recombinant truncated polypeptide.
  • Trypsin did not cleave the Lys-Glu bond of residues 26 and 27 in experiments conducted on the recombinant GLP1 polypeptide. While not in any way meant to limit the invention, it is believed that cleavage at residues 26 and 27 by trypsin is not favored because of the presence of the acidic glutamic acid residue.
  • the cleavage with trypsin is conducted in the presence of a glycyl biotin nucleophiles so that the cleavage of the Ala-Phe-Ala leaving unit is followed by the addition of glycyl biotin.
  • the biotinylated GLP1 derivative can be bound to an immobilized avidin.
  • the resulting column can be used for an affinity probe for membrane bound GLP receptors .

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Abstract

Système de fixation à une protéine d'un marqueur, d'un support ou d'un agent bioactif, au niveau d'un site de troncature au sein de la protéine. Plus spécialement, il s'agit d'un procédé de fixation d'un nucléophile d'acide aminé, de dérivé d'acide aminé, de peptide ou de polypeptide, à un noyau protéique obtenu à partir d'une protéine du noyau et d'un groupe labile. Selon un mode de réalisation, on utilise une endopeptidase pour fixer un nucléophile marqué à un noyau protéique tel qu'un anticorps. Selon d'autres modes de réalisation, on met en ÷uvre un procédé de fixation d'un noyau protéique à un support d'immobilisation, et d'un procédé de fixation d'un agent bioactif à un noyau protéique.
PCT/US1994/008127 1993-07-20 1994-07-19 Procede d'endomodification de proteins WO1995003321A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000053624A1 (fr) * 1999-03-11 2000-09-14 Gryphon Sciences Synthese chimique et utilisation de domaines de recepteurs de proteines membranaires solubles
WO2001045745A3 (fr) * 1999-12-21 2002-05-10 Acambis Res Ltd Technologie de liaison reversible pour conjugaison controlee
EP1350846A4 (fr) * 2000-12-07 2005-01-26 Univ Keio Proteine a c-terminaison modifiee et procede permettant d'obtenir cette proteine, agent de modification et matrice de traduction necessaire a la production d'une proteine a c-terminaison modifiee, et procede de detection de l'interaction proteique avec l'utilisation de ladite proteine
EP2256133A1 (fr) * 1997-01-08 2010-12-01 Sigma-Aldrich Co. Bio-conjugaison de macromolécules

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0359399A1 (fr) * 1988-08-12 1990-03-21 Carlbiotech Ltd. A/S Procédé de production enzymatique des dipeptides
EP0360433A1 (fr) * 1988-08-26 1990-03-28 Robin Ewart Offord Dérivés de protéines et procédé pour leur préparation
WO1991000296A1 (fr) * 1989-06-30 1991-01-10 Board Of Regents Of The University Of Nebraska Matieres bioactives de liaison enzymatique a des proteines
EP0490249A1 (fr) * 1990-12-10 1992-06-17 F. Hoffmann-La Roche Ag Procédé de préparation enzymatique de GRF(1-44)NH2

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0359399A1 (fr) * 1988-08-12 1990-03-21 Carlbiotech Ltd. A/S Procédé de production enzymatique des dipeptides
EP0360433A1 (fr) * 1988-08-26 1990-03-28 Robin Ewart Offord Dérivés de protéines et procédé pour leur préparation
WO1991000296A1 (fr) * 1989-06-30 1991-01-10 Board Of Regents Of The University Of Nebraska Matieres bioactives de liaison enzymatique a des proteines
EP0490249A1 (fr) * 1990-12-10 1992-06-17 F. Hoffmann-La Roche Ag Procédé de préparation enzymatique de GRF(1-44)NH2

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2256133A1 (fr) * 1997-01-08 2010-12-01 Sigma-Aldrich Co. Bio-conjugaison de macromolécules
WO2000053624A1 (fr) * 1999-03-11 2000-09-14 Gryphon Sciences Synthese chimique et utilisation de domaines de recepteurs de proteines membranaires solubles
JP2002539136A (ja) * 1999-03-11 2002-11-19 グリフォン サイエンシーズ 可溶性膜タンパク質受容体ドメイン類の化学合成と使用
WO2001045745A3 (fr) * 1999-12-21 2002-05-10 Acambis Res Ltd Technologie de liaison reversible pour conjugaison controlee
EP1350846A4 (fr) * 2000-12-07 2005-01-26 Univ Keio Proteine a c-terminaison modifiee et procede permettant d'obtenir cette proteine, agent de modification et matrice de traduction necessaire a la production d'une proteine a c-terminaison modifiee, et procede de detection de l'interaction proteique avec l'utilisation de ladite proteine
US7150978B2 (en) 2000-12-07 2006-12-19 Keio University Recombinant template used for producing a carboxy-terminal modified protien and a method of producing a carboxy-terminal modified protein

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