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WO2003056004A1 - Ameliorations de la thermolabilite d'enzymes - Google Patents

Ameliorations de la thermolabilite d'enzymes Download PDF

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Publication number
WO2003056004A1
WO2003056004A1 PCT/AU2002/001488 AU0201488W WO03056004A1 WO 2003056004 A1 WO2003056004 A1 WO 2003056004A1 AU 0201488 W AU0201488 W AU 0201488W WO 03056004 A1 WO03056004 A1 WO 03056004A1
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Prior art keywords
alkaline phosphatase
amino acid
group
argininamide
residue
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PCT/AU2002/001488
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English (en)
Inventor
Khawar Sohail Siddiqui
Ricardo Cavicchioli
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Unisearch Limited
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Priority to AU2002335940A priority Critical patent/AU2002335940A1/en
Publication of WO2003056004A1 publication Critical patent/WO2003056004A1/fr

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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)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • 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
    • C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
    • C12Y301/03001—Alkaline phosphatase (3.1.3.1)

Definitions

  • the invention relates to chemical modification of alkaline phosphatase for improving the ther olability of the enzyme.
  • the invention also provides a modified alkaline phosphatase produced by this method.
  • Alkaline phosphatases are widespread in nature and catalyse the hydrolysis of phosphate ester bonds at alkaline pH. Alkaline phosphatases have many applications in biological research and in medical diagnostics. Alkaline phosphatases are used, for example, in molecular biology to catalyse the hydrolysis of a 5 phosphate group from a nucleic acid molecule. This prevents the 5' ends of a linear nucleic acid molecule that comprises complementary nucleotide sequence from forming a phosphodiester bond with the hydroxy1 group at the 3' end of a DNA molecule in a ligation reaction.
  • An alkaline phosphatase must be removed or inactivated prior to a ligation reaction to prevent the phosphatase from catalysing the hydrolysis of a 5' phosphate group for forming a phosphodiester bond from a further nucleic acid molecule that is to be phosphodiester -bonded, or in other words, ligated, with a de- phosphorylated linear nucleic acid molecule.
  • the dephosphorylated linear nucleic acid molecules are precluded from ligating with the further nucleic acid molecules.
  • SAP Shrimp Alkaline Phosphatase
  • SAP can be inactivated by heating, however, it must be heated to at least 65°C to permit inactivation of the phosphatase. Prolonged heating at greater than 50°C may cause DNA to denature and/or other enzymes in the reaction to inactivate. This may affect the overall yield of ligated DNA molecules .
  • alkaline phosphatases that have a thermolability that is sufficient to permit inactivation at a temperature that is lower than that of known alkaline phosphatases, and preferably lower than the temperature at which SAP is inactivated.
  • thermolability of the enzyme is improved, or in other words, thermolability sufficient for permitting inactivation of the enzyme at a temperature that is lower than the temperature at which alkaline phosphatase not having a glycinamide, ethylendiamine, argininamide, amidino, diethylenetriamine pentaacetic or aromatic group linked to a side chain of an amino acid residue, and/or to a terminal amino acid residue, is inactivated.
  • the invention provides a modified alkaline phosphatase comprising at least one group selected from the group consisting of glycinamide, ethylendiamine, argininamide, amidino, diethylenetriamine pentaacetic and aromatic, the glycinamide, ethylendiamine, argininamide, amidino, diethylenetriamine pentaacetic or aromatic group being linked to a side chain of an amino acid residue of the phosphatase and/or to a terminal amino acid residue of the phosphatase.
  • the modified alkaline phosphatase is inactivated at a temperature that is lower than the temperature at which the corresponding unmodified alkaline phosphatase is inactivated.
  • the aromatic group is a derivative of benzene.
  • the aromatic group is an optionally substituted phenylalkylamino group, an optionally substituted aralkylamino group or an optionally substituted benzoyl group.
  • the aromatic group is selected from the group consisting of benzylamine, aniline, benzoic acid, phthalic acid, mellitic acid, succinic acid, sulfanilic acid and pyromellitic acid.
  • the aromatic group may be a heterocyclic or homocyclic six membered ring, a heterocyclic five membered ring or a cofused aromatic ring.
  • the aromatic group is a heterocyclic amine.
  • the heterocyclic amine is pyridine .
  • the glycinamide, ethylendiamine, argininamide or aromatic group is linked to the carboxyl group of a side chain of an amino acid, and/or to the carboxy-terminal amino acid.
  • the glycinamide, ethylenediamine, argininamide or aromatic group is linked to the side chain of a glutamate residue, an aspartate residue, and/or to a carboxy-terminal amino acid residue.
  • the diethylenetriamine pentaacetic, amidino or aromatic group is linked to the amino group of a side chain of an amino acid and/or to an amino terminal amino acid.
  • the glycinamide, ethylendiamine, argininamide or aromatic group is linked to the side chain of an aspartate, glutamate and/or to the carboxy-terminal amino acid of the alkaline phosphatase.
  • the amidino, diethylenetriamine pentaacetic or aromatic group is linked to the side chain of a lysine residue and/or to the amino- terminal amino acid of the alkaline phosphatase.
  • the glycinamide, ethylendiamine, argininamide, amidino, diethylenetriamine pentaacetic or aromatic group is linked to the side chain of an amino acid or to a terminal amino acid by an amide bond.
  • the enzyme comprises at least one glycinamide and at least one ethylenediamine residue, at least one of the residues being linked to a side chain of an amino acid residue of the enzyme.
  • any alkaline phosphatase may be modified by the procedures described herein.
  • the alkaline phosphatase before modification is isolated from an organism selected from the group consisting of vertebrates, invertebrates, angiosperms, fungi, yeast, bacteria, archeae and algae.
  • the organism is a psychrophilic or a mesophilic organism.
  • the organism is a mammal or a shrimp.
  • the shrimp is P. borealia .
  • the alkaline phosphatase before modification may be produced by recombinant means wherein a nucleic acid molecule isolated from the group consisting of vertebrates, invertebrates, angiosperms, fungi, yeast, bacteria, archeae and algae is transformed into a recipient organism.
  • the organism is a psychrophilic or a mesophilic organism.
  • the organism is a mammal or a shrimp.
  • the shrimp is P. Jborealis.
  • the alkaline phosphatase has the amino acid sequence of SAP or CIAP. More preferably, the alkaline phosphatase has the amino acid sequence of the SAP encoded by the sequence deposited under Genbank accession no. AJ296089 or biologically active fragment or variant thereof.
  • the invention provides a process for modifying alkaline phosphatase comprising the step of contacting an alkaline phosphatase with a compound which comprises a molecule selected from the group consisting of glycinamide, ethylendiamine, argininamide, amidino, diethylenetriamine pentaacetic and aromatic, so that the glycinamide, ethylendiamine, argininamide, amidino, diethylenetriamine pentaacetic or aromatic group is linked to a side chain of an amino acid residue of the alkaline phosphatase and/or to a terminal amino acid residue of the alkaline phosphatase.
  • a compound which comprises a molecule selected from the group consisting of glycinamide, ethylendiamine, argininamide, amidino, diethylenetriamine pentaacetic and aromatic, so that the glycinamide, ethylendiamine, argininamide, amid
  • the process comprises activating carboxyl groups of amino acid residues of the alkaline phosphatase in the presence of a glycinamide nucleophile, an ethylendiamine nucleophile, an argininamide nucleophile or an aromatic nucleophile.
  • a glycinamide nucleophile is glycinamide hydrochloride.
  • the ethylendiamine nucleophile is ethylenediamine dihydrochloride.
  • the argininamide nucleophile is argininamide dihydrochloride.
  • the aromatic nucleophile is selected from the group consisting of pyridine hydrochloride, benzylamine hydrochloride, sodium sulphanilic acid and aniline hydrochloride.
  • the carboxyl groups may be activated by any compound that provides sufficient conditions for a glycinamide, an ethylendiamine, an argininamide, or an aromatic group to be linked to the side chain of an amino acid residue of the enzyme, or linked to the carboxyl terminal amino acid residue of the enzyme.
  • carboxyl groups are activated by carbodiimide.
  • the carboxyl groups of the enzyme are activated by 1- ethyl-3 (3-dimethylaminopropyl) carbodiimide or l-(3- dimethylaminopropyl) -3 -ethyl carbodiimide methiodide.
  • the process comprises contacting the enzyme with an aromatic anhydride in conditions sufficient for linking an aromatic group to an amino group of an amino acid residue of the enzyme, or to the amino terminal amino acid residue of the enzyme.
  • the aromatic anhydride may be any aromatic containing anhydride.
  • the aromatic anhydride is selected from the group consisting of benzoic anhydride, pyromellitic dianhydride, mellitic trianhydride, trimellitic anhydride, succinic anhydride and phthalic anhydride .
  • the process comprises contacting the enzyme with diethylenetriamine pentacetic dianhydride in conditions sufficient for linking a diethylenetriamine pentacetic acid group to an amino group of an amino acid residue of the enzyme, or to the amino terminal amino acid residue of the enzyme.
  • the process comprises contacting the enzyme with O-methylisourea or 3,5- dimethylpyrazole-1-carboxamidine nitrate in conditions sufficient for linking an amidino group to an amino group of an amino acid residue of the enzyme.
  • the amidino group is linked to an amino group of lysine to convert the lysine residue to a homoarginine residue.
  • the process comprises the further step of contacting the enzyme with an agent for controlling the linkage of the glycinamide, ethylendiamine, argininamide, amidino, diethylenetriamine pentaacetic or aromatic group to a side chain of an amino acid residue and/or to a terminal amino acid residue located in a catalytic site of the enzyme.
  • the agent is a substrate or an inhibitor of the enzyme.
  • the inhibitor is ATP, NADH, K 2 HP0 4 or Na 3 P ⁇ 4/K2HP ⁇ 4/Na2HP ⁇ 4/KH2P0 4 /NaH2P ⁇ 4.
  • the invention provides a modified alkaline phosphatase produced by the process of the second aspect of the invention.
  • the invention provides a composition comprising a modified alkaline phosphatase according to the above described aspects of the invention and a suitable carrier.
  • the carrier comprises 25mM Sodium Acetate/Acetic Acid, pH5.5 and ImM MgCl 2 and
  • Such a composition is useful for maintaining the stability of the enzyme at 4°C, for example, when the composition is stored.
  • the invention provides a method of cleaving the 5' phosphate from a nucleic acid molecule comprising the step of exposing a nucleic acid molecule having a 5' phosphate to the modified alkaline phosphatase according to the invention.
  • modified alkaline phosphatase which is thermolabile compared to the corresponding unmodified alkaline phosphatase.
  • modified alkaline phosphatase refers to any alkaline phosphatase having a glycinamide, ethylendiamine, argininamide, amidino, diethylenetriamine pentaacetic or aromatic group linked to a side chain of an amino acid and/or to a terminal amino acid.
  • corresponding unmodified alkaline phosphatase refers to an alkaline phosphatase having the same amino acid sequence as the modified alkaline phosphatase but not having a glycinamide, ethylendiamine, argininamide, amidino, diethylenetriamine pentaacetic or aromatic group linked to a side chain of an amino acid and/or to a terminal amino acid of the alkaline phosphatase.
  • glycinamide, ethylendiamine, argininamide, amidino, diethylenetriamine pentaacetic or aromatic groups were linked to the side chains of amino acid residues of shrimp alkaline phosphatase (SAP) , or linked to the terminal amino acid residue of SAP, and the resultant enzyme was observed to have a decreased half- life (t ⁇ /2 ) , relative to t ⁇ /2 of unmodified SAP, at a temperature above which the optimal activity of SAP is observed.
  • SAP shrimp alkaline phosphatase
  • a modified alkaline phosphatase comprising a glycinamide residue linked to a side chain of an amino acid residue of the alkaline phosphatase, or linked to the terminal amino acid residue of the alkaline phosphatase, was observed to have t ⁇ 2 at 60°C that is approximately 3 fold lower than t ⁇ 2 of unmodified SAP at 60°C.
  • a further advantage of a modified alkaline phosphatase of the invention is that the temperature at which the modified alkaline phosphatase has maximal activity (T opt ) is typically decreased relative to the T opt of the corresponding unmodified alkaline phosphatase.
  • T opt the temperature at which the modified alkaline phosphatase has maximal activity
  • a modified alkaline phosphatase comprising an ethylenediamine residue linked to a side chain of an amino acid residue of the enzyme and/or linked to the terminal amino acid residue of the enzyme, was observed to have a T o p t of 34°C, as compared with the T opt of the corresponding unmodified alkaline phosphatase, which was observed to be 41°C.
  • the maximal rate of dephosphorylation catalysed by the modified alkaline phosphatase is typically observed at a temperature lower than the temperature for maximal activity of the corresponding alkaline phosphatase.
  • a further advantage of the modified alkaline phosphatase of the invention is that it has decreased stability compared with the stability of the corresponding unmodified alkaline phosphatase, in acidic pH conditions.
  • a modified alkaline phosphatase has a decreased half-life, as compared with the half-life of the corresponding unmodified alkaline phosphatase, at pH 5.5. This advantageously provides an additional or alternative approach for inactivation of a modified alkaline phosphatase of the invention.
  • the first step in preparing the modified alkaline phosphatase of the invention involves selecting the alkaline phosphatase to which the glycinamide, ethylendiamine, argininamide, amidino, diethylenetriamine pentaacetic or aromatic group is to be linked.
  • alkaline phosphatases are classified according to their ability to cleave phosphate. While alkaline phosphatases may be isolated from different organisms and therefore have slightly different activities and/or properties, the overall classification is the same. In other words, an alkaline phospahatase isolated from one organisms will have very similar properties to an alkaline phospatase isolated from a different organism.
  • the "unmodified" alkaline phospahatase can be "wild-type", “naturally-occurring” or “recombinant” alkaline phosphatase or variant thereof obtained from any suitable origin, such as vertebrate, invertebrate, shrimp, angiosperm, fungus, yeast, prokaryotes including bacteria, archeaebacteria and eubacteria, or a mesophilic organism. Origin can further be psychrotolerant, psychrotrophic, mesophilic or extremophilic (psychrophilic, psychrotrophic, thermophilic, barophilic, alkalophilic, acidophilic, halophilic, etc.).
  • mutants of wild-type alkaline phosphatases are mutants of wild-type alkaline phosphatases. Mutants can be obtained eg. by protein and/or genetic engineering, chemical and/or physical modifications of wild-type enzymes. Common practice as well is the expression of the enzyme via host organisms in which the genetic material responsible for the production of the enzyme has been cloned. Examples of organisms from which the alkaline phosphatase may be obtained include shrimp or other Crustacea, bacteria including Escherichia coli , Bacillus subtilis, Bacillus lichenoformis, Arachniotus sp.
  • amino acids sensitive to oxidation or amino acids that affect the surface charges are of interest.
  • the isoelectric point of such enzymes may also be modified by the substitution of some charged amino acids, eg. an increase in isoelectric point may help to improve compatibility with anionic surfactants.
  • the stability of the enzymes may be further enhanced by the creation of eg. additional salt bridges and enforcing metal binding sites to increase chelant stability.
  • amino acid refers to any of the naturally occurring amino acids, as well as optical isomers (enantiomers and diastereomers) , synthetic analogs and derivatives thereof.
  • ⁇ -Amino acids comprise a carbon atom to which is bonded an amino group, a carboxyl group, a hydrogen atom, and a distinctive group referred to as a "side chain.”
  • ⁇ -Amino acids also comprise a carbon atom to which is bonded an amino group, a carboxyl group, and two distinctive groups (which can be the same group or can be different groups) , in which case the amino acid has two side chains.
  • side chains of naturally occurring amino acids include, for example, hydrogen (eg., as in glycine) , alkyl (eg., as in alanine, valine, leucine, isoleucine) , substituted alkyl (eg., as in threonine, serine, methionine, cysteine, aspartic acid, asparagine, glutamic acid, glutamine, arginine, and lysine), arylalkyl (eg., as in phenylalanine) , substituted arylalkyl (eg., as in tyrosine) , selenocysteine, pyrolysine and heteroarylalkyl (eg., as in histidine and tryptophan) .
  • hydrogen eg., as in glycine
  • alkyl eg., as in alanine, valine, leucine, isoleucine
  • substituted alkyl eg.
  • amino acid also includes ⁇ - ⁇ -, ⁇ -, and ⁇ -amino acids, and the like, and ⁇ -imino acids such as proline.
  • amino acids includes proline.
  • Non-naturally occurring amino acids are also known in the art, as set forth in, for example, Williams (ed.), "Synthesis of Optically Active ⁇ -Amino Acids", Pergamon Press, 1989; Evans et al . (1990) J. Amer. Chem. Soc, 112:4011-4030; Pu et al . (1991) J. Amer. Chem. Soc . 56:1280-1283; and Williams et al . (1991) J. Amer. Chem. Soc. 113:9276-9286; and all references cited therein.
  • mutants or variants of the unmodified enzymes encompassed in the present invention may be prepared by introducing appropriate nucleotide changes into the DNA or cDNA of the unmodified enzyme and thereafter expressing the resulting modified DNA or cDNA in a host cell, or by in vitro synthesis.
  • Such mutant and/or variants include, for example, deletions from, or insertions or substitutions of, amino acid residues within the amino acid sequence of the unmodified enzyme. Any combination of deletion, insertion, and substitution may be made to arrive at an amino acid sequence variant of the unmodified enzyme, provided that such variant possesses the desired characteristics described herein.
  • nucleotide sequence of nucleic acid molecules which encode alkaline phosphatase that would be particularly useful in the present invention are part of the public domain.
  • Nucleic acid molecules which encode alkaline phosphatase may be found, for example, in the Genbank database (www.ncbi.nlm.gov/entrez) under Genbank accession number AJ296089, NM_013059, AF285233, NM_141058, NM 007431, AB055428, AB013386, M14169, XM_044131,
  • amino acid sequence variants of the unmodified enzyme There are two principal variables in the construction of amino acid sequence variants of the unmodified enzyme: the location of the mutation site and the nature of the mutation. These are variants from the amino acid sequence of the unmodified enzyme, and may represent naturally occurring allelic forms of the unmodified enzyme, or predetermined mutant forms of the unmodified enzyme made by mutating the unmodified enzyme DNA, either to arrive at an allele or a variant not found in nature. In general, the location and nature of the mutation chosen will depend upon the unmodified enzyme characteristic to be modified.
  • mutations can be made in the unmodified enzyme nucleotide sequence without affecting the amino acid sequence of the unmodified enzyme encoded thereby.
  • Other mutations can be made that will result in the unmodified enzyme having an amino acid sequence that is very different, but which is functionally active.
  • Such functionally active amino acid sequence variants of the unmodified enzyme are selected, for example, by substituting one or more amino acid residues with other amino acid residues of a similar or different polarity or charge .
  • Insertional, deletional, and substitutional changes in the amino acid sequence of the unmodified enzyme may be made to improve the stability of the unmodified enzyme before it is used in the present invention.
  • trypsin or other protease cleavage sites are identified by inspection of the encoded amino acid sequence for an arginyl or lysinyl residue. These are rendered inactive to protease by substituting the residue with another residue, preferably a basic residue such as glutamine or a hydrophobic residue such as serine; by deleting the residue; or by inserting a prolyl residue immediately after the residue.
  • any cysteine residues not involved in maintaining the proper conformation of the unmodified enzyme for functional activity may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking.
  • Cysteinyl residues most commonly are reacted with ⁇ - haloacetates (and corresponding amines) , such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues also are derivatized by reaction with bromotrifluoroacetone, ⁇ -bromo- ⁇ - (5-imidozoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro- 2-pyridyl disulfide, methyl 2-pyridyl disulfide, p- chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-l, 3-diazole.
  • Histidyl residues are derivatized by reaction with diethylpyrocarbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain.
  • Para- bromophenacyl bromide also is useful; the reaction is preferably performed in 0.1M sodium cacodylate at pH 6.0. Lysinyl and amino terminal residues are reacted with succinic or other carboxylic acid anhydrides.
  • Derivatization with these agents has the effect of reversing the charge of the lysinyl residues.
  • suitable reagents for derivatizing ⁇ -amino-containing residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; 0- ethylisourea; 2, 4-pentanedione; and transaminase- catalyzed reaction with glyoxylate.
  • Arginyl residues are modified by reaction with one or several conventional reagents, among them phenylglyoxal,
  • Plasmids are DNA molecules that are capable of replicating within a host cell, either extrachromosomally or as part of the host cell chromosome (s) , and are designated by a lower case "p" preceded and/or followed by capital letters and/or numbers .
  • DNA is cleaved into fragments, tailored, and ligated together in the form desired to generate the vectors required. Normally it is desirable to add a signal sequence which provides for secretion of the enzyme.
  • useful genes are: 1) Signal sequence-- (pro-peptide) --carbohydrate- binding domain- -linker-- enzyme sequence of interest, or 2) Signal sequence-- (pro-peptide) --enzyme sequence of interest- -linker-- carbohydrate-binding domain, in which the pro-peptide sequence normally contains 5-100, eg. 5- 25, amino acid residues.
  • plasmids or vectors capable of expressing enzymes having the amino acid sequences derived from fragments of more than one polypeptide is well known in the art (see, for example, WO 90/00609 and WO 95/16782) .
  • the DNA of the enzyme of interest may be included within a replication system for episomal maintenance in an appropriate cellular host or may be provided without a replication system, where it may become integrated into the host genome.
  • the DNA may be introduced) into the host in accordance with known techniques such as transformation, transfection, microinjection or the like.
  • Host cells that are transformed or transfected with the above-described plasmids and expression vectors are cultured in conventional nutrient media modified as is appropriate for inducing promoters or selecting for drug resistance or some other selectable marker or phenotype.
  • the culture conditions such as temperature, pH, and the like, suitably are those previously used for culturing the host cell used for cloning or expression, as the case may be, and will be apparent to those skilled in the art.
  • Suitable host cells for cloning or expressing the vectors herein are prokaryotes, yeasts, and higher eukaryotes, including insect, vertebrate, and mammalian host cells.
  • Suitable prokaryotes include eubacteria, such as Gram-negative or Gram-positive organisms, for example, E. coli , Bacillus species such as B. subtilis, Pseudomonas species such as P. aeruginosa , Salmonella typhimurium, or Serratia marcescens .
  • eubacteria such as Gram-negative or Gram-positive organisms, for example, E. coli , Bacillus species such as B. subtilis, Pseudomonas species such as P. aeruginosa , Salmonella typhimurium, or Serratia marcescens .
  • eukaryotic microbes such as filamentous fungi or yeast are suitable hosts for enzyme-encoding vectors.
  • Saccharomyces cerevisiae or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms.
  • a number of other genera, species, and strains are commonly available and useful herein, such as Schizosaccharomyces pombe, Beach and Nurse, Nature 290:140-142 (1981), Pichia pastoris, Cregg, et al . , Bio/Technology 5:479-485 (1987); Sreekrishna, et al .
  • Suitable host cells for the expression of mutant, variant or wild-type enzymes are also derived from multicellular organisms. Such host cells are capable of complex processing and glycosylation activities. In principle, any higher eukaryotic cell culture is useable, whether from vertebrate or invertebrate culture. It will be appreciated, however, that because of the species-, tissue-, and cell-specificity of glycosylation, Rademacher, et al . , Ann. Rev. Biochem . 57:785-838 (1988), the extent or pattern of glycosylation of an enzyme of interest in a foreign host cell typically will differ from that of the enzyme obtained from a cell in which it is naturally expressed.
  • invertebrate cells include insect cells .
  • Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can be utilized as hosts.
  • plant cells are transfected by incubation with certain strains of the bacterium Agrobacter-ium tumefaciens, which has been previously altered to contain mutant, variant or wild-type enzyme DNA.
  • Agrobacter-ium tumefaciens the DNA encoding the mutant, variant or wild-type enzyme is transferred into cells, such that they become transfected, and will, under appropriate conditions, express the mutant, variant or wild-type enzyme.
  • D ⁇ A segments isolated from the upstream region of the T- D ⁇ A 780 gene are capable of activating or increasing transcription levels of plant-expressible genes in recombinant D ⁇ A-containing plant tissue.
  • European Pat. Pub. No. EP 321,196 published June 21, 1989).
  • monkey kidney cells (CV1, ATCC CCL 70) ; African green monkey kidney cells (VERO-76, ATCC CRL-1587) ; human cervical carcinoma cells (HELA, ATCC CCL 2) ; canine kidney cells (MDCK, ATCC CCL 34) ; buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51) ; TRI cells (Mather, et al . , Annals N. Y. Acad. Sci . 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2) .
  • the host may be grown to express the enzyme.
  • One particularly preferred system of expression useful in this invention involves fermentation in which the mutant, variant or wild-type enzyme of interest is introduced into a bacterial or yeast host as described above and then cultured in the presence of nutrient media containing suitable carbon and nitrogen sources and inorganic salts, using procedures known in the art such as that described in Bennett, J.W. and LaSure(Eds.) "More Gene Manipulations in Fungi", Academic Press, CA, (1991) . Temperature ranges and other conditions suitable for growth and production of enzymes are also known in the art and are described in for example, Bailey, J.E. and Ollis, D.F., "Biochemical Engineering Fundamentals", McGraw-Hill Book Company, NY, 1986.
  • fermentation refers to any growth condition which results in production of an enzyme by an organism(s) . It will be understood by persons skilled in the art that fermentation can refer to small or large scale fermentation and includes, for example, shake- flask cultivation, continuous, batch, fed-batch and solid state fermentation in laboratory or industrial fermenters.
  • the mutant, variant or wild-type alkaline phosphatase may be isolated by any method that is suitable for isolating active alkaline phosphatase from organisms and/or growth media. Suitable methods known in the art include, for example, centrifugation, filtration, spray drying, evaporation, precipitation, ion exchange chromatography, gel filtration chromatography, hydrophobic-interaction chromatography (HIC) , affinity chromatography or the like, and combinations thereof. An example of an isolation method is provided in US Patent No. 5,434,067.
  • the alkaline phosphatase for use in the method of the invention may be a single isolated alkaline phosphatase or a mixture of alkaline phosphatases from the same or different sources. Purified shrimp alkaline phosphatase and purified calf intestinal phosphatase are commercially available from, for example, Roche-Boerhinger Mannheim or Fermentas .
  • the alkaline phosphatase is used as a single isolated alkaline phosphatase.
  • the alkaline phosphatase may represent part of a mixture of enzymes or other compounds.
  • the alkaline phosphatase may be used in a crude form with contaminating compounds including other enzymes and proteins.
  • alkaline phosphatase is obtained as described above, a glycinamide, ethylendiamine, argininamide, amidino, diethylenetriamine pentaacetic or aromatic group is contacted with the amino acid side chains of the enzyme.
  • the term "contacted" refers to sufficient contact between the amino acid side chain and the glycinamide, ethylendiamine, argininamide, amidino, diethylenetriamine pentaacetic or aromatic group which permits the glycinamide, ethylendiamine, argininamide, amidino, diethylenetriamine pentaacetic or aromatic group to be linked to the amino acid side chain in conditions sufficient for linking the glycinamide, ethylendiamine, argininamide, amidino, diethylenetriamine pentaacetic or aromatic group to an amino acid side chain and/or terminal amino acid of the enzyme.
  • the term "linked” refers to any linkage formed between a portion of the amino acid side chain and the glycinamide, ethylendiamine, argininamide, amidino, diethylenetriamine pentaacetic or aromatic group. It will be appreciated by those skilled in the art that following linkage of the glycinamide, ethylendiamine, argininamide, amidino, diethylenetriamine pentaacetic or aromatic group to the amino acid side chain, the amino acid side chain to which the glycinamide, ethylendiamine, argininamide, amidino, diethylenetriamine pentaacetic or aromatic group is linked will be altered and will differ from the amino acid side chains common to many proteins owing to the presence of the glycinamide, ethylendiamine, argininamide, amidino, diethylenetriamine pentaacetic or aromatic group linked to the side chain of the amino acid.
  • amino acid side chains “common to many proteins” will be understood by those skilled in the art to mean the side chains belonging to the amino acids alanine, asparagine, aspartate, arginine, cysteine, glutamine, glutamate, glycine, histidine, isoleucine, lysine, leucine, methionine, phenylalanine, proline, serine, tyrosine, tryptophan, threonine and valine.
  • the glycinamide, ethylendiamine, argininamide, amidino, diethylenetriamine pentaacetic or aromatic group may be linked to the amino acid side chain in any manner.
  • the glycinamide, ethylendiamine, argininamide, amidino, diethylenetriamine pentaacetic or aromatic group is linked to the amino acid side chain through one or more nitrogen atoms.
  • the glycinamide, ethylendiamine, argininamide, amidino, diethylenetriamine pentaacetic or aromatic group is linked to the amino acid side chain through an amide bond.
  • the glycinamide, ethylendiamine, argininamide, amidino, diethylenetriamine pentaacetic or aromatic group may be linked to the amino acid side chain through a linker.
  • a "linker” is a molecule which is not part of the glycinamide, ethylendiamine, argininamide, amidino, diethylenetriamine pentaacetic or aromatic group nor part of the amino acid side chain, but serves to link the glycinamide, ethylendiamine, argininamide, amidino, diethylenetriamine pentaacetic or aromatic group to the side chain of the amino acid.
  • the "conditions sufficient" for linking the glycinamide, ethylendiamine, argininamide, amidino, diethylenetriamine pentaacetic or aromatic group to a side chain of an amino acid residue or a terminal amino acid residue may be any conditions which allow a reaction to occur between the amino acid side chain and the glycinamide, ethylendiamine, argininamide, amidino, diethylenetriamine pentaacetic or aromatic group which results in linkage of the glycinamide, ethylendiamine, argininamide, amidino, diethylenetriamine pentaacetic or aromatic group to the amino acid side chain.
  • the conditions sufficient for linking the glycinamide, ethylendiamine, argininamide or aromatic group to a side chain of an amino acid residue and/or a terminal amino acid residue comprise activating the carboxyl groups of the alkaline phosphatase at a temperature preferably between 18°C and 50°C, more preferably between 20°C and 40°C, even more preferably between 20°C and 28°C, and a pH preferably between 3.0 and 7.0, more preferably between 4.5 and pH7.0, and contacting the activated carboxyl groups with a glycinamide nucleophile, an ethylenediamine nucleophile, an argininamide nucleophile or an aromatic nucleophile.
  • the carboxyl groups are on the side chains of aspartate and/or glutamate residues and/or on the carboxy- terminal amino acid.
  • the term "activated" refers to a modification of an existing functional group to generate or introduce a new reactive functional group from the prior existing functional group, wherein the new reactive functional group is capable of undergoing reaction with another functional group to form a covalent bond.
  • a component containing carboxylic acid (-COOH) groups can be activated by reaction with N-hydroxy- succinimide or N-hydroxysulfosuccinimide using known procedures, to form an activated carboxylate (which is a reactive electrophilic group), ie., an N- hydroxysuccinimide ester or an N-hydroxysulfosuccinimide ester, respectively.
  • Activation of carboxylic acids may be accomplished in a variety of ways and by using a number of different reagents as described in Larock, "Comprehensive Organic Transformations", VCH Publishers, New York, 1989, all of which are incorporated herein by reference. However, activation often involves reaction with a suitable hydroxyl-containing compound in the presence of a dehydrating agent such as dicyclohexylcarbodiimide (DCC) or dicyclohexylurea (DHU) .
  • a dehydrating agent such as dicyclohexylcarbodiimide (DCC) or dicyclohexylurea (DHU) .
  • a carboxylic acid can be reacted with an alkoxy-substituted N-hydroxy- succinimide or N-hydroxysulfosuccinimide in the presence of DCC to form reactive electrophilic groups, the N- hydroxysuccinimide ester and the N-hydroxysulfosuccinimide ester, respectively.
  • Carboxylic acids may also be activated by reaction with an acyl halide such as an acyl chloride (eg., acetyl chloride), again using known procedures, to provide an activated electrophilic group in the form of a reactive anhydride group.
  • a carboxylic acid may be converted to an acid chloride group using, eg., thionyl chloride or an acyl chloride capable of an exchange reaction.
  • thionyl chloride or an acyl chloride capable of an exchange reaction Specific reagents and procedures used to carry out such activation reactions will be known to those of ordinary skill in the art and are described in the pertinent texts and literature.
  • activated carboxyl groups means the rendering of one or more the carboxyl groups of the side chains of an amino acid of an enzyme reactive with a nucleophile.
  • nucleophile and “nucleophilic” refer to a functional group that is electron rich, has an unshared pair of electrons acting as a reactive site, and reacts with a positively charged or electron-deficient site, generally present on another molecule .
  • electrophilic and “electrophilic” refer to a functional group that is susceptible to nucleophilic attack, ie., susceptible to reaction with an incoming nucleophilic group.
  • Electrophilic groups herein are positively charged or electron-deficient, typically electron-deficient .
  • carboxyl groups of the enzyme are activated by incubating the enzyme with a carbodiimide, preferably utilising the carbodiimide condensation method described by Sheehan and Hess, and Khorana [Sheehan and Hess, J. Am. Chem. Soc. 77:1067, 1955; Khorana, Chem.Ind. 1087, 1995].
  • carboxyl groups of the enzyme are activated by incubating the enzyme with Woodwards reagent.
  • the difference between carbodiimide and Woodwards Reagent activation of carboxyl group is that in case of carbodiimide the carboxyl group must be protonated (COOH) , whereas in case of Woodwards reagent the carboxyl group may be ionised (COO " ) .
  • COOH carboxyl group
  • Woodwards reagent the carboxyl group may be ionised
  • the reaction is a condensation of the carboxyl with a substituted carbodiimide to form an active O-acylourea intermediate.
  • Nucleophilic substitution with the amine containing group forms a stable amide with elimination of the substituted urea.
  • the carbodiimide may be, for example, l-ethyl-3(3- dimethylaminopropy1) carbodiimide or 1- (3- dimethylaminopropyl) -3-ethyl carbodiimide methipdide.
  • Methods for the use of carbodiimide in the activation of carboxyl groups are provided in, for example, Carraway, K.L. and Koshland, D.E. Jr, Carbodiimide modification of proteins. In: Methods in Enzymology (Hirs, C.H.W. and Timasheff, S.N., Eds.) Academic Press, New York, 1972, XXV, 616 - 623 .
  • glycinamide, ethylenediamine, argininamide or aromatic nucleophile refers to any nucleophile comprising a glycinamide, ethylenediamine, argininamide or aromatic molecule.
  • the glycinamide nucleophile is glycinamide hydrochloride
  • the ethylenediamine nucleophile is ethylenediamine dihydrochloride
  • the argininamide nucleophile is argininamide dihydrochloride
  • the aromatic nucleophile is selected from the group consisting of aniline hydrochloride, benzylamine hydrochloride, sodium sulfanilic acid and pyridine hydrochloride .
  • the carboxyl groups of the amino acid may be activated with carbodiimide prior to adding the nucleophile to the reaction.
  • the carboxyl groups of the amino acid side chains are activated with carbodiimide in the presence of the nucleophile.
  • the nucleophile is dissolved in an appropriate buffer such as, for example, K 2 HP ⁇ 4 /KH 2 P0 4 buffer at a pH of preferably between 3.0 and 7.0, more preferably between 4.0 and 6.0.
  • the buffer may optionally contain an alkaline phosphatase inhibitor.
  • Suitable inhibitors may be ATP, NADH or a 3 P04/K2HP04/Na 2 HP04/KH2P04/NaH 2 P04, or any other substrate of alkaline phosphatase which is capable of protecting the active site of alkaline phosphatase from linkage of a glycinamide, ethylenediamine, argininamide or aromatic molecule to the active site residues.
  • Alkaline phosphatase is added to the solution either as a dried preparation or as a solution.
  • the reaction is initiated by the addition of carbodiimide to a final concentration of preferably between 30mM and 200mM, more preferably between 40mM and lOOmM.
  • the modified alkaline phosphatase comprises:
  • a diethylenetriamine pentaacetic may be linked to the amino group of a side chain of an amino acid or to the amino terminus of the alkaline phosphatase by contacting the enzyme with a diethylenetriamine pentaacetic dianhydride.
  • An aromatic group may be linked to the amino group of a side chain of an amino acid or to the amino terminus of the alkaline phosphatase by contacting the enzyme with an aromatic anhydride.
  • the amino group of an amino acid side chain is the amino group of the side chain of lysine residues.
  • an aromatic anhydride is an anhydride which comprises an aromatic group.
  • Aromatic anhydrides may include, for example, benzoic anhydride, pryromellitic dianhydride, mellitic trianhydride, trimellitic anhydride, succinic anhydride and phthalic anhydride.
  • the alkaline phosphatase is dissolved or diluted in a buffer, preferably between pH 7.0 and 12, more preferably between pH 8.0 and 11.0.
  • a alkaline phosphatase inhibitor may be included as mentioned above.
  • the diethylenetriamine pentaacetic dianhydride or aromatic anhydride is preferably added to the enzyme solution to begin the reaction. The resulting solution is thereafter incubated for an amount of time that can readily be determined by those skilled in the art.
  • the diethylenetriamine pentaacetic dianhydride or aromatic anhydride may be added to the enzyme solution in a single application or as a plurality of smaller aliquots.
  • the invention provides a modified alkaline phosphatase comprising:
  • Benzenetetracarboxylic acid residue linked to an amino acid side chain of a lysine residue of the phosphatase, and/or to an amino terminal amino acid; (d) at least one diethylenetriamine pentaacetic acid residue linked to an amino acid side chain of a lysine residue of the phosphatase, and/or to an amino terminal amino acid of the phosphatase.
  • an amidino group may be linked to the amino group of the side chain of a lysine residue by contacting the alkaline phosphatase with O- methylisourea or 3, 5-dimethylpyrozole-l-carboxamidine nitrate under conditions sufficient to permit conversion of a lysine residue of the enzyme to a homoarginine residue.
  • modified alkaline phosphatase comprising two or more different aromatic groups linked to side chains of amino acids of the enzyme.
  • the aromatic groups may be linked, for example, by incubating the alkaline phosphatase with carbodiimide in the presence of two or more different aromatic nucleophiles, or by incubating the enzyme in, for example, the presence of two or more different aromatic anhydrides .
  • the modified alkaline phosphatase may comprise an aromatic group linked to a carboxyl group and an aromatic group linked to an amino group.
  • both of the above reactions may be applied to the alkaline phosphatase.
  • the alkaline phosphatase may be reacted with a carbodiimide and an aromatic nucleophile, and subsequently reacted with an aromatic anhydride.
  • the alkaline phosphatase may be reacted with an aromatic anhydride followed by reaction with a carbodiimide and an aromatic nucleophile.
  • the invention provides a modified alkaline phosphatase comprising: (a) at least one aniline residue linked to an amino acid side chain of a glutamate residue or aspartate residue of the phosphatase, and/or to the carboxyl terminal residue of the phosphatase, and at least one benzoic acid residue linked to an amino acid side chain of a lysine residue of the phosphatase;
  • the modified alkaline phosphatase may comprise a glycinamide, ethylendiamine, argininamide or aromatic group linked to a carboxyl group and an amidino, diethylenetriamine pentaacetic or aromatic group linked to an amino group.
  • the enzyme may be reacted with a carbodiimide and nucleophile, and subsequently reacted with an anhydride.
  • the enzyme may be reacted with an anhydride followed by reaction with a carbodiimide and a nucleophile.
  • the modified alkaline phosphatase comprises:
  • the modified alkaline phosphatase may be used directly after the glycinamide, ethylendiamine, argininamide, amidino, diethylenetriamine pentaacetic or aromatic group has been linked to the enzyme, in one preferred embodiment the modified alkaline phosphatase is purified using a conventional enzyme purification method.
  • the modified enzymes of the present invention may be purified by salting out with ammonium sulfate or other salts, gel filtration, dialysis, ion exchange chromatography, hydrophobic chromatography, crystallization, or by using a solvent such as acetone or an alcohol or the like. All of these methods are disclosed in well known literature such as Inman, "Methods in Enzymology", Vol. 34, "Affinity Techniques, Enzyme
  • glycinamide, ethylendiamine, argininamide, amidino, diethylenetriamine pentaacetic or aromatic groups have been linked to the side chain of an amino acid residue or terminal amino acid of the enzyme
  • assays well known in the art may be employed.
  • the linking glycinamide, ethylendiamine, argininamide, amidino, diethylenetriamine pentaacetic or aromatic group may be readily "observed” using techniques such as For example, structures from X- ray crystallographic techniques, NMR techniques, de novo modelling, homology modelling, PAGE, amino acid analysis, et cetera .
  • compositions comprising the modified alkaline phosphatase according to the present invention.
  • the compositions comprise the modified alkaline phosphatase according to the invention as the major enzymatic component.
  • the composition may optionally comprise one or more additional enzymes, such as a proteolytic enzyme, a ribonuclease, a DNA or RNA polymerase, an Rnase inhibitor, a topoisomerase, a ligase, a nuclease or a terminal transferase.
  • the composition may also comprise one or more nucleic acid molecules such as primers or polynucleotides, vectors etc.
  • the composition may be prepared in accordance with methods known in the art and may be in the form of a liquid or a dry composition.
  • the composition may be in a powder form.
  • the additional enzymes to be included in the composition may be stabilized in accordance with methods known in the art.
  • the amount of the polypeptide composition of the invention to be used, and the conditions under which the composition is used, may be determined on the basis of methods known in the art, and will depend on the purpose for which the composition is used.
  • the enzyme according to the present invention and compositions comprising the enzyme may be applied in processes conventionally involving the action of alkaline phosphatases.
  • Major applications for alkaline phosphatases are found in molecular biology.
  • the modified alkaline phosphatase of the present invention may be used to treat double stranded DNA ends to remove 5' phosphate groups prior to ligation with phosphorylated DNA.
  • the modified alkaline phosphatase may be used in the non-isotopic detection of proteins and nucleic acids in combination with, for example, chromogenic and other substrates such as 5-bromo-4-chloro-3-indolyl phosphate (X-phos) , para-nitrophenyl phosphate, 4-methylumbelliferyl phosphate (4-MUP) , 3- (2' -spiroadamantane) -4-methoxy-4- (3' ' -phosphoryloxy)phenyl-l,2-dioxetane (AMPPD) or ( ⁇ - naphthyl phosphate.
  • X-phos 5-bromo-4-chloro-3-indolyl phosphate
  • 4-MUP 4-methylumbellife
  • Example 1 Carboxyl group modification of alkaline phosphatase (AP) general methodology 1. Prepare a nucleophilic solution comprising a glycinamide, ethylenediamine, argininamide or aromatic nucleophile, to a final concentration in the range of 25 mM -1.25 M.
  • AP alkaline phosphatase
  • nucleophilic solution for example a competitive inhibitor or non-competitive inhibitor, or mixture thereof, to protect active-site residues of AP during the modification reaction described below.
  • an inhibitor of AP for example a competitive inhibitor or non-competitive inhibitor, or mixture thereof, to protect active-site residues of AP during the modification reaction described below.
  • substrate for AP to protect active-site residues during modification.
  • Adjust the pH of the nucleophilic solution to 3.0-5.5 depending upon the number of carboxyls to be modified and the pKa of the amino group of the nucleophile used.
  • Example 2 Modification of SAP with aniline SAP was purchased from Roche.
  • a nucleophilic solution was prepared by dissolving Aniline.HCl to a final concentration of (25 mM) in ⁇ 1.5 ml water.
  • Concentrated KH 2 PO 4 /K 2 HPO 4 , pH 5.5 buffer was added to a final concentration of 20 mM to the nucleophilic solution (P0 4 ⁇ 3 acts as a competitive inhibitor) .
  • the pH was finally adjusted to ⁇ 5.5 with 2 M NaOH and the volume made up to 2.5 ml.
  • 1 ml of nucleophilic solution was taken and 5-10 ⁇ l (5- 10 Units or 0.9-1.8 ⁇ g) SAP (Roche) was added.
  • the reaction was initiated by adding solid 0.01 g of 1-Ethyl- 3- (3-Dimethylaminopropyl) Carbodiimide.HCl (-50 mM) .
  • Example 3 Modification of SAP with benzylamine.
  • a nucleophilic solution was prepared by dissolving Benzylamine.HCl to a final concentration of 250 mM in ⁇ 1.5 ml water. Concentrated KH 2 PO 4 /K 2 HPO 4 , pH 5.5 buffer was added to a final concentration of 20 mM to the nucleophilic solution (PO 4 "3 acts as a competitive inhibitor). The pH was finally adjusted to - 5.5 with 2 M NaOH and the volume made up to 2.5 ml. 1 ml of nucleophilic solution was taken and 5-10 ⁇ l (5- 10 Units or 0.9-1.8 ⁇ g) of SAP (purchased from Roche) was added.
  • the reaction was initiated by adding solid 0.01 g of 1-Ethyl- 3- (3-Dimethylaminopropyl) Carbodiimide.HCl ( ⁇ 50 mM) . After a specified time (15 - 30 min) , the reaction was stopped by adding 1 ml of 100 mM sodium acetate, pH 7 buffer followed by 1 ml of 1.5 M hydroxylamine.HCl, pH 7 for the regeneration of tyrosines for 6-14 hours. The modified enzyme was then thoroughly dialyzed against 20 mM glycine/NaOH, pH 7.6 buffer to remove reagents. The modified enzyme was then characterized or alternatively, subjected further to amino-group modification, as described below. The t ⁇ /2 and T opt values are shown in Table 1.
  • a nucleophilic solution was prepared by dissolving Pyridine.HCl to a final concentration of 25 mM in ⁇ 1.5 ml water. Concentrated KH 2 PO4/K 2 HPO 4 , pH 5.5 buffer was added to a final concentration of 20 mM to the nucleophilic solution (PO 4 "3 acts as a competitive inhibitor) . The pH was finally adjusted to ⁇ 5.5 with 2 M NaOH and the volume made up to 2.5 ml. 1 ml of nucleophilic solution was taken and 5-10 ⁇ l (5- 10 Units or 0.9-1.8 ⁇ g) of SAP (purchased from Roche) was added.
  • the reaction was initiated by adding solid 0.01 g of l-Ethyl-3- (3- Dimethylaminopropyl) Carbodiimide.HCl ( ⁇ 50 mM) . After a specified time (15 - 30 min) , the reaction was stopped by adding 1 ml of 100 mM sodium acetate, pH 7 buffer followed by 1 ml of 1.5 M hydroxylamine.HCl, pH 7 for the regeneration of tyrosines for 6-14 hours. The modified enzyme was then thoroughly dialyzed against 20 mM glycine/NaOH, pH 7.6 buffer to remove reagents. The modified enzyme was then characterized or alternatively, subjected further to amino-group modification, as described below. The t ⁇ 2 and T opt values are shown in Table 1.
  • Example 5 Modification of SAP with glycinamide
  • a nucleophilic solution was prepared by dissolving Glycinamide.HCl to a final concentration of 1M in - 1.5 ml water. Concentrated KH 2 PO 4 /K 2 HPO 4 , pH 5.5 buffer was added to a final concentration of 20 mM to the nucleophilic solution (PO 4 "3 acts as a competitive inhibitor) . The pH was finally adjusted to ⁇ 5.5 with 2 M NaOH and the volume made up to 2.5 ml. 1 ml of nucleophilic solution was taken and 5-10 ⁇ l (5- 10 Units or 0.9-1.8 ⁇ g) of SAP (purchased from Roche) was added. The reaction was initiated by adding solid 0.01 g of l-Ethyl-3- (3-
  • Example 6 Modification of SAP with argininamide.
  • a nucleophilic solution was prepared by dissolving Argininamide .2HC1 to a final concentration of 1M in ⁇ 1.5 ml water. Concentrated KH 2 PO 4 /K 2 HPO 4 , pH 5.5 buffer was added to a final concentration of 20 mM to the nucleophilic solution (P0 4 "3 acts as a competitive inhibitor) . The pH was finally adjusted to - 5.5 with 2 M NaOH and the volume made up to 2.5 ml. 1 ml of nucleophilic solution was taken and 5-10 ⁇ l (5- 10 Units or 0.9-1.8 ⁇ g) of SAP (purchased from Roche) was added.
  • the reaction was initiated by adding solid 0.01 g of 1-Ethyl- 3- (3-Dimethylaminopropyl) Carbodiimide.HCl (-50 mM) . After a specified time (15 - 30 min) , the reaction was stopped by adding 1 ml of 100 mM sodium acetate, pH 7 buffer followed by 1 ml of 1.5 M hydroxylamine.HCl, pH 7 for the regeneration of tyrosines for 6-14 hours. The modified enzyme was then thoroughly dialyzed against 20 mM glycine/NaOH, pH 7.6 buffer to remove reagents. The modified enzyme was then characterized or alternatively, subjected further to amino-group modification, as described below. The t ⁇ 2 and T opt values are shown in Table 1.
  • Example 7 Modification of SAP with ethylenediamine.
  • a nucleophilic solution was prepared by dissolving
  • Ethylenediamine .2HC1 to a final concentration of (1M) in - 1.5 ml water.
  • Concentrated KH 2 PO 4 /K 2 HPO 4 , pH 5.5 buffer was added to a final concentration of 20 mM to the nucleophilic solution (P0 4 ⁇ 3 acts as a competitive inhibitor).
  • the pH was finally adjusted to - 5.5 with 2 M NaOH and the volume made up to 2.5 ml. 1 ml of nucleophilic solution was taken and 5-10 ⁇ l (5- 10 Units or 0.9-1.8 ⁇ g) of SAP (purchased from Roche) was added.
  • the reaction was initiated by adding solid 0.01 g of 1-Ethyl- 3- (3-Dimethylaminopropyl) Carbodiimide.HCl (-50 mM) .
  • Example 8 Amino group modification of alkaline phosphatase (AP) : general methodology.
  • step 4. Dialyze the mixture of step 4. against appropriate buffer to remove reagents.
  • SAP (purchased from Roche) was diluted (1 ⁇ l/ml buffer) in 100 mM K 2 HPO 4 /KH 2 PO 4 , pH 8.3 buffer containing 100 mM sodium acetate.
  • equal volumes of carboxyl-group modified enzyme and 200 mM K 2 HPO 4 /KH 2 PO 4 , pH 8.3 buffer containing 200 mM sodium acetate were mixed.
  • 1M benzoic anhydride solution in dimethylsulfoxide solvent was prepared. The reaction was initiated by adding 25 ⁇ l of anhydride solution to 4 ml of enzyme solution. Further aliquots of 25 ⁇ l anhydride solution were added for further modification.
  • the t ⁇ 2 and T opt values for SAP modified with phthalic anhydride are shown in Table 2.
  • the t ⁇ /2 and T opt values for SAP modified with phthalic anhydride and aniline, and phthalic anhydride and argininamide, are shown in Table 3.
  • Example 11 Modification of SAP with succinic anhydride.
  • SAP purchased from Roche
  • equal volumes of carboxyl-group modified enzyme and 200 mM K 2 HPO 4 /KH 2 PO4, pH 8.3 buffer containing 200 mM sodium acetate were mixed.
  • 1M succinic anhydride solution in dimethylsulfoxide solvent was prepared. The reaction was initiated by adding 25 ⁇ l of anhydride solution to 4 ml of enzyme solution.
  • SAP (purchased from Roche) was diluted (1 ⁇ l/ml buffer) in 100 mM K 2 HP0 4 /KH 2 P0 4 , pH 8.3 buffer containing 100 mM sodium acetate.
  • equal volumes of carboxyl-group modified enzyme and 200 mM K 2 HP0 4 /KH 2 PO 4 , pH 8.3 buffer containing 200 mM sodium acetate were mixed.
  • 1M pyromellitic anhydride solution in dimethylsulfoxide solvent was prepared. The reaction was initiated by adding 25 ⁇ l of anhydride solution to 4 ml of enzyme solution. Further aliquots of 25 ⁇ l anhydride solution were added for further modification. After appropriate time (30 -60 min) , the modified enzyme was dialyzed against 20 mM glycine/NaOH, pH 7.6 buffer to remove reagents .
  • SAP (purchased from Roche) was dialysed against water to remove free amino groups. 0.5 to lmg of the dialysed SAP was then added to 1ml of a 0.5M solution of 3,5- dimethyl pyrazole-1-carboxamidine nitrate pH9.5. The reaction was incubated at 2°C for 72hrs, after which the enzyme was dialysed against 50mM sodium acetate/acetic acid buffer or KH 2 P0 4 /citric acid, pH5 buffer.
  • a nucleophilic solution was prepared by dissolving sodium sulfanilic acid to a final concentration of (lOOmM) in - 1.5 ml water.
  • Concentrated KH 2 PO 4 /K 2 HPO 4 , pH 5.2 buffer was added to a final concentration of 40 mM to the nucleophilic solution (PO 4 "3 acts as a competitive inhibitor) together with ImM MgS0 4 .
  • the pH was finally adjusted to - 5.2 with 2 M NaOH and the volume made up to 2.5 ml. 1 ml of nucleophilic solution was taken and 5-10 ⁇ l (5- 10 Units or 0.9-1.8 ⁇ g) of SAP (purchased from Roche) was added.
  • the reaction was initiated by adding solid 0.01 g of l-Ethyl-3- (3-Dimethylaminopropyl) Carbodiimide.HCl (50 mM) . After a specified time (15 - 30 min) , the reaction was stopped by adding 1 ml of 100 mM sodium acetate, pH 7 buffer. The modified enzyme was then thoroughly dialyzed against 50 mM glycine/NaOH, pH 8.5 buffer to remove reagents. The modified enzyme was then characterized or alternatively, subjected further to amino-group modification, as described below. The t ⁇ /2 and T opt values are shown in Table 4.
  • Example 15 Modification of SAP with diethylenetriamine pentaacetic dianhydride.
  • SAP (purchased from Roche) was diluted (1 ⁇ l/ml buffer) in borate buffer pH 8.6 or 10.2 (as indicated in Table 4). Solid diethylenetriamine pentaacetic dianhydride was added to the enzyme solution to a final concentration as shown in Table 4. After an appropriate time (30 -60 min), the modified enzyme was dialyzed against 50 mM glycine/NaOH, pH 8.5 buffer to remove reagents.
  • Example 16 Assay of alkaline phosphatase activity of carboxyl group- and amino group -modified enzymes.
  • Assay Mixture 6 mM p-nitrophenyl phosphate (Substrate) was made in 100 mM Glycine/NaOH, pH 10.4 buffer containing 0.1 mM MgCl 2 and 0.1 mM ZnCl 2 .
  • Assay Procedure 0.5 ml of Assay mixture was taken in an eppendorf tube, incubated at 37 °C. The reaction was started by adding 10-50 ⁇ l of SAP or SAP modified according to any one of Examples 1 to 13 above. After 15 min the reaction was terminated by adding 0.5 ml of 2M NaOH solution. A 405 was read against the reagent blank.
  • Optimum temperature T opt values were determined by assaying SAP or SAP modified according to any one of Examples 1 to 15 above, as described above, at various temperatures. The results are shown in accompanying Tables 1 to 4.

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Abstract

Cette invention concerne une phosphatase alcaline modifiée comprenant au moins un groupe sélectionné parmi les groupes glycinamide, éthylènediamine, argininamide, amidino, diéthylènetriamine pentaacétate et aromatique, lesquels groupes glycinamide, éthylènediamine, argininamide, amidino, diéthylènetriamine pentaacétate ou aromatique étant liés à une chaîne latérale d'un résidu aminoacide de la phosphatase et/ou à un résidu aminoacide terminal de la phosphatase, laquelle modification permet d'obtenir une enzyme à thermostabilité réduite. Cette invention concerne également des procédés de modification d'une phosphatase alcaline.
PCT/AU2002/001488 2001-12-21 2002-11-01 Ameliorations de la thermolabilite d'enzymes WO2003056004A1 (fr)

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PCT/AU2002/001488 WO2003056004A1 (fr) 2001-12-21 2002-11-01 Ameliorations de la thermolabilite d'enzymes
PCT/AU2002/001484 WO2003056000A1 (fr) 2001-12-21 2002-11-01 Enzyme modifiee et procede de modification
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WO2019069977A1 (fr) * 2017-10-03 2019-04-11 キッコーマン株式会社 Procédé pour la production de phosphatase alcaline, phosphatase alcaline obtenue au moyen dudit procédé, et vecteur et transformant pour sa production
JPWO2019069977A1 (ja) * 2017-10-03 2020-12-24 キッコーマン株式会社 アルカリホスファターゼの製造方法及びそれを用いて得られるアルカリホスファターゼ、並びにその製造のためのベクター及び形質転換体
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JP7692683B2 (ja) 2017-10-03 2025-06-16 キッコーマン株式会社 アルカリホスファターゼの製造方法及びそれを用いて得られるアルカリホスファターゼ、並びにその製造のためのベクター及び形質転換体

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WO2003056002A1 (fr) 2003-07-10
WO2003055999A1 (fr) 2003-07-10

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