EP0904384A2 - Tyrosine-phosphatase-related protein - Google Patents

Tyrosine-phosphatase-related protein

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Publication number
EP0904384A2
EP0904384A2 EP97918050A EP97918050A EP0904384A2 EP 0904384 A2 EP0904384 A2 EP 0904384A2 EP 97918050 A EP97918050 A EP 97918050A EP 97918050 A EP97918050 A EP 97918050A EP 0904384 A2 EP0904384 A2 EP 0904384A2
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EP
European Patent Office
Prior art keywords
dna
protein
cell differentiation
tyrosine
immunization
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP97918050A
Other languages
German (de)
French (fr)
Inventor
Annemarie Poustka
Petra Kioschis
Jocelyn Laporte
Ling Jia Hu
Jean Louis Institut de Génétique et de MANDEL
Niklas Dahl
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Deutsches Krebsforschungszentrum DKFZ
Institut National de la Sante et de la Recherche Medicale INSERM
Original Assignee
Deutsches Krebsforschungszentrum DKFZ
Institut National de la Sante et de la Recherche Medicale INSERM
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Application filed by Deutsches Krebsforschungszentrum DKFZ, Institut National de la Sante et de la Recherche Medicale INSERM filed Critical Deutsches Krebsforschungszentrum DKFZ
Publication of EP0904384A2 publication Critical patent/EP0904384A2/en
Withdrawn legal-status Critical Current

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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)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to a tyrosine-phosphatase-related protein, such a coding DNA and a method for producing such.
  • the invention further relates to the use of the DNA and the protein and antibodies directed against the protein.
  • Tyrosine kinase and tyrosine phosphatase are enzymes that work in opposite directions. Tyrosine kinase causes the phosphorylation of certain tyrosine residues in proteins, while tyrosine phosphatase reverses this phosphorylation. Both enzymes play an important role in signal transduction, control of cell growth and in cell differentiation.
  • myotubular myopathy This is an X-linked disease with altered muscle cells. The disease manifests itself in general muscle weakness, in particular spontaneous movements are hardly possible. Breathing in newborns is also severely restricted. This often leads to premature death. However, the causes of the impaired cell differentiation in myotubular myopathy are not known.
  • the present invention is therefore based on the object of providing a means with which the causes of disorders in cell differentiation, in particular in myotubular myopathy, can be examined and, if appropriate, treated. According to the invention, this is achieved by the subject matter in the claims.
  • the present invention thus relates to a tyrosine-phosphatase-related protein, the protein comprising the amino acid sequence of FIG. 1 or an amino acid sequence different therefrom by one or more amino acids.
  • the present invention is based on the knowledge of the applicant that in
  • a protein exists which has homologies to known tyrosine phosphatases and also a tyrosine phosphatase activity, but which differs from known tyrosine phosphatases at the DNA level by hybridization under customary conditions differs.
  • Such a protein has the amino acid sequence of FIG. 1 or an amino acid sequence that differs therefrom by one or more amino acids.
  • the applicant has also recognized that the protein is important for cell differentiation. He found that this protein in a truncated or mutated form, i.e. without or only with limited tyrosine phosphatase activity, leads to disorders in the differentiation of cells, in particular muscle cells, and very particularly to the formation of myotubular myopathy.
  • TRP thyroid-related protein
  • Another object of the present invention is a nucleic acid coding for (TVP).
  • This can be an RNA or a DNA.
  • the latter can e.g. be a genomic DNA or a cDNA.
  • a DNA is preferred which comprises the following:
  • hybridizing DNA indicates a DNA that is commonly used
  • the DNA of FIG. 1 was deposited with the DSM (German Collection of Microorganisms and Cell Cultures) as hp6 under DSM 10558 on March 4, 996.
  • a DNA according to the invention is described below in the form of a cDNA. This is an example of every DNA covered by the present invention.
  • cosmid library which comprises the region Xq28 of the human genome.
  • a cosmid library is e.g. the Xq28-specific cosmid library (cf. Kioschis, P. et al., Cytogenet. Cell. Genet. 58, (1 991), 2070), which was produced from the cell hybrid QIZ (cf. Warren, ST et al., Proc Natl. Acad. Sci. USA 87 (1990), 3856-3860). From this become the cosmid clones
  • a cDNA according to the invention can be present in a vector or expression vector.
  • examples of such are known to the person skilled in the art.
  • these are, for example, pGEMEX, pUC derivatives, pGEX-2T, pET3b and pQE-8, the latter being preferred.
  • yeast Examples include pY100 and Ycpad i, while pKCR, pEFBOS, cDM8 and pCEV4 must be specified for expression in animal cells.
  • the baculovirus expression vector pAcSGHisNT-A is particularly suitable for expression in insect cells.
  • suitable cells in order to express a cDNA according to the invention which is present in an expression vector.
  • suitable cells include the E. coli strains HB101, DH1, x1 776, JM101, JM 109, BL21 and SG 1 3009, the latter being preferred, the yeast strain Saccharomyces cerevisiae and the animal cells L, 3T3, FM3A, CHO , COS, Vero and HeLa and the insect cells sf9.
  • a cDNA according to the invention has to be inserted into an expression vector. He is also aware that this DNA can be inserted in conjunction with a DNA coding for another protein or peptide, so that the cDNA according to the invention can be expressed in the form of a fusion protein.
  • Another object of the present invention is an antibody directed against a protruding protein or fusion protein.
  • Such an antibody can be produced by conventional methods. It can be polyclonal or monoclonal. For its production, it is favorable to immunize animals, in particular rabbits or chickens for a polyclonal and mice for a monoclonal antibody, with an above (fusion) protein or fragments thereof. Further “boosters" of the animals can be carried out with the same (fusion protein or fragments thereof. The polyclonal antibody can then be obtained from the serum or egg yolk of the animals. For the monoclonal antibody, animal spleen cells are fused with myeloma cells.
  • the present invention makes it possible to investigate the causes of cell differentiation disorders, particularly in muscle cells and very particularly in myotubular myopathy.
  • a nucleic acid according to the invention in particular a DNA, and primers derived therefrom, it can be determined in mammals, in particular humans, whether they contain and / or express a gene which codes for a truncated or mutated (TVP) in the above sense .
  • TVP truncated or mutated
  • kits which contains the above nucleic acid, in particular DNA, and / or primers derived therefrom, as well as carriers and customary auxiliaries.
  • an inventive (TVP) can be introduced into mammals, especially humans.
  • TVP a protein that is not considered foreign by the respective body, e.g. Transferrin or BSA to couple.
  • a nucleic acid according to the invention in particular a DNA, can also be introduced into mammals, in particular humans, and expressed there.
  • the expression of (TVP) can be controlled and regulated with an antibody according to the invention.
  • the present invention thus makes a major contribution to the diagnostic and therapeutic detection of disorders of cell differentiation, in particular in muscle cells and very particularly in myotubular myopathy. valley.
  • Example 1 Production and cleaning of an inventive (TVP)
  • the DNA of FIG. 1 was used as a template to produce an inventive (TVP).
  • a PCR procedure was carried out. The following was used as a primer pair:
  • MTM-F 5'-CAGGGATCCGATGGCAGCCGAGCAGCCTGGCAAC-3 'and MTM-R: 5'-GGGGGATCCTCAGAAGTGAGTTTGCACATGGGG-3'
  • the amplified DNA was in each case cleaved with Bam HI and inserted into the expression vector pQE-8 (Diagen) cleaved with Barn HI.
  • the expression plasmid pQ / TVP was obtained.
  • pQ / TVP was used to transform E.coli SG 13009 (cf. Gottesman, S. et al., J. Bacteriol. 148 y (1981), 265-273).
  • the bacteria were cultured in an LB medium with 100 g / ml ampicillin and 25 ⁇ g / ml kanamycin and induced for 4 h with 60 ⁇ M isopropyl- ⁇ -D-thiogalactopyranoside (IPTG). A 6 M guanidine hydrochloride was added
  • a chromatography (Ni-NTA resin) was carried out with the lysate in the presence of 8 M urea in accordance with the manufacturer's (Diagen) instructions for the chromatography material.
  • the bound fusion protein was eluted in a pH 3.5 buffer. After neutralization, the fusion protein was an 18% SDS polyacrylamide
  • Example 2 Production and detection of an antibody according to the invention
  • a fusion protein according to the invention from Example 1 was subjected to 1 8% SDS-polyacrylamide gel electrophoresis. After staining the gel with 4 M sodium acetate, an approximately 205 kD band was cut out of the gel and incubated in phosphate-buffered saline. Pieces of gel were sedimented before the protein concentration of the supernatant was determined by SDS-polyacrylamide gel electrophoresis followed by a Coomassie blue stain. Animals were immunized with the gel-purified fusion protein as follows:
  • 35 ⁇ g of gel-purified fusion protein in 0.7 ml of PBS and 0.7 ml of complete or incomplete Freund's adjuvant were used for each immunization.
  • the rabbit serum was tested in the immunoblot.
  • a fusion protein according to the invention from Example 1 was subjected to SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose filter (cf. Khyse-Andersen, J., J. Biochem. Biophys. Meth. 10, (1 984), 203-209).
  • the nitrocellulose filter cf. Khyse-Andersen, J., J. Biochem. Biophys. Meth. 10, (1 984), 203-209).
  • Antibodies were extracted from egg yolk and tested in a Western blot. Polyclonal antibodies according to the invention have been detected.

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Abstract

The present invention concerns a tyrosine-phosphatase-related protein, a DNA which codes for such a protein and a process for producing such a protein. Also disclosed is the use of the DNA and protein and antibodies directed against the protein.

Description

Tyrosin-Phosphatase-verwandtes Protein Tyrosine phosphatase-related protein
Die vorliegende Erfindung betrifft ein Tyrosin-Phosphatase-verwandtes Protein, eine ein solches kodierende DNA und ein Verfahren zur Herstellung eines sol¬ chen. Ferner betrifft die Erfindung die Verwendung der DNA und des Proteins sowie gegen das Protein gerichtete Antikörper.The present invention relates to a tyrosine-phosphatase-related protein, such a coding DNA and a method for producing such. The invention further relates to the use of the DNA and the protein and antibodies directed against the protein.
Tyrosin-Kinase und Tyrosin-Phosphatase sind Enzyme, die entgegengesetzt wirken. Tyrosin-Kinase bewirkt die Phosphorylierung bestimmter Tyrosin-Reste in Proteinen, während Tyrosin-Phosphatase diese Phosphorylierung wieder rückgängig macht. Beide Enzyme spielen eine wichtige Rolle in der Signaltrans- duktion, der Kontrolle des Zellwachstums und in der Zelldifferenzierung.Tyrosine kinase and tyrosine phosphatase are enzymes that work in opposite directions. Tyrosine kinase causes the phosphorylation of certain tyrosine residues in proteins, while tyrosine phosphatase reverses this phosphorylation. Both enzymes play an important role in signal transduction, control of cell growth and in cell differentiation.
Es sind Erkrankungen bekannt, die auf Störungen in der Zelldifferenzierung beruhen. Eine dieser Erkrankungen ist myotubuläre Myopathie. Dies ist eine X- chromosomal-gebundene Erkrankung, bei der veränderte Muskelzellen vorliegen. Die Erkrankung äußert sich in allgemeiner Muskelschwäche, insbesondere sind spontane Bewegungen kaum möglich. Ebenso ist die Atmung bei Neugeborenen stark eingeschränkt. Dies führt vielfach zum frühzeitigen Tod . Die Ursachen für die gestörte Zelldifferenzierung bei myotubulärer Myopathie sind allerdings nicht bekannt.Diseases are known which are based on disturbances in cell differentiation. One of these diseases is myotubular myopathy. This is an X-linked disease with altered muscle cells. The disease manifests itself in general muscle weakness, in particular spontaneous movements are hardly possible. Breathing in newborns is also severely restricted. This often leads to premature death. However, the causes of the impaired cell differentiation in myotubular myopathy are not known.
Der vorliegenden Erfindung liegt somit die Aufgabe zugrunde, ein Mittel bereitzu¬ stellen, mit dem Störungen in der Zelldifferenzierung, insbesondere bei myotubu¬ lärer Myopathie, in ihrer Ursache untersucht und gegebenenfalls therapiert werden können. Erfindungsgemäß wird dies durch die Gegenstände in den Patentansprüchen erreicht.The present invention is therefore based on the object of providing a means with which the causes of disorders in cell differentiation, in particular in myotubular myopathy, can be examined and, if appropriate, treated. According to the invention, this is achieved by the subject matter in the claims.
Gegenstand der vorliegenden Erfindung ist somit ein Tyrosin-Phosphatase- verwandtes Protein, wobei das Protein die Aminosäuresequenz von Fig. 1 oder eine hiervon durch eine oder mehrere Aminosäuren unterschiedliche Aminosäu¬ resequenz umfaßt.The present invention thus relates to a tyrosine-phosphatase-related protein, the protein comprising the amino acid sequence of FIG. 1 or an amino acid sequence different therefrom by one or more amino acids.
Die vorliegende Erfindung beruht auf der Erkenntnis des Anmelders, daß inThe present invention is based on the knowledge of the applicant that in
Tieren, besonders Säugetieren, ganz besonders dem Menschen, ein Protein existiert, das Homologien zu bekannten Tyrosin-Phosphatasen und auch eine Tyrosin-Phosphatase-Aktivität aufweist, sich aber von bekannten Tyrosin- Phosphatasen auf dem DNA-Level durch Hybridisierung unter üblichen Bedingun- gen unterscheidet. Ein solches Protein weist die Aminosäuresequenz von Fig. 1 oder eine hiervon durch eine oder mehrere Aminsoäuren unterschiedliche Amino¬ säuresequenz auf. Ferner hat der Anmelder erkannt, daß das Protein wichtig für die Zelldifferenzierung ist. Er hat gefunden, daß dieses Protein in verkürzter bzw. mutierter Form, d.h. ohne oder nur mit eingeschränkter Tyrosin-Phosphatase- Aktivität, zu Störungen der Differenzierung von Zellen, insbesondere Muskelzel¬ len, und ganz besonders zur Ausbildung von myotubulärer Myopathie, führt.Animals, especially mammals, very particularly humans, a protein exists which has homologies to known tyrosine phosphatases and also a tyrosine phosphatase activity, but which differs from known tyrosine phosphatases at the DNA level by hybridization under customary conditions differs. Such a protein has the amino acid sequence of FIG. 1 or an amino acid sequence that differs therefrom by one or more amino acids. The applicant has also recognized that the protein is important for cell differentiation. He found that this protein in a truncated or mutated form, i.e. without or only with limited tyrosine phosphatase activity, leads to disorders in the differentiation of cells, in particular muscle cells, and very particularly to the formation of myotubular myopathy.
In der vorliegenden Erfindung wird vorstehendes Protein mit "Tyrosin-verwand- tes Protein" (TVP) bezeichnet.In the present invention, the above protein is called "tyrosine-related protein" (TVP).
Ein weiterer Gegenstand der vorliegenden Erfindung ist eine für (TVP) kodie¬ rende Nukleinsäure. Dies kann eine RNA oder eine DNA sein. Letztere kann z.B. eine genomische DNA oder eine cDNA sein. Bevorzugt ist eine DNA, die folgen¬ des umfaßt:Another object of the present invention is a nucleic acid coding for (TVP). This can be an RNA or a DNA. The latter can e.g. be a genomic DNA or a cDNA. A DNA is preferred which comprises the following:
(a) die DNA von Fig. 1 oder eine hiervon durch ein oder mehrere Basenpaare unterschiedliche DNA, (b) eine mit der DNA von (a) hybridisierende DNA, oder(a) the DNA of FIG. 1 or a DNA different therefrom by one or more base pairs, (b) a DNA hybridizing with the DNA of (a), or
(c) eine mit der DNA von (a) oder (b) über den degenerierten genetischen Code verwandte DNA.(c) a DNA related to the DNA of (a) or (b) via the degenerate genetic code.
Der Ausdruck "hybridisierende DNA" weist auf eine DNA hin, die unter üblichenThe term "hybridizing DNA" indicates a DNA that is commonly used
Bedingungen, insbesondere bei 20°C unter dem Schmelzpunkt der DNA, mit einer DNA von (a) hybridisiert.Conditions, in particular at 20 ° C. below the melting point of the DNA, hybridized with a DNA from (a).
Die DNA von Fig. 1 wurde bei der DSM (Deutsche Sammlung von Mikroorganis- men und Zellkulturen) als hp6 unter DSM 10558 am 4. März 1 996 hinterlegt.The DNA of FIG. 1 was deposited with the DSM (German Collection of Microorganisms and Cell Cultures) as hp6 under DSM 10558 on March 4, 996.
Nachstehend wird eine erfindungsgemäße DNA in Form einer cDNA beschrieben. Diese steht beispielhaft für jede unter die vorliegende Erfindung fallende DNA.A DNA according to the invention is described below in the form of a cDNA. This is an example of every DNA covered by the present invention.
Zur Herstellung einer erfindungsgemäßen cDNA ist es günstig, von einer Cos- mid-Bibliothek auszugehen, welche die Region Xq28 des menschlichen Genoms umfaßt. Eine solche Cosmid-Bibliothek ist z.B. die Xq28 spezifische Cosmidbi- bliothek (vgl. Kioschis, P. et al., Cytogenet. Cell. Genet. 58, (1 991 ), 2070) die aus dem Zellhybrid QIZ hergestellt wurde (vgl. Warren, S.T. et al., Proc. Natl. Acad. Sei. USA 87 (1990), 3856-3860). Von dieser werden die Cosmid-KloneTo produce a cDNA according to the invention, it is favorable to start from a cosmid library which comprises the region Xq28 of the human genome. Such a cosmid library is e.g. the Xq28-specific cosmid library (cf. Kioschis, P. et al., Cytogenet. Cell. Genet. 58, (1 991), 2070), which was produced from the cell hybrid QIZ (cf. Warren, ST et al., Proc Natl. Acad. Sci. USA 87 (1990), 3856-3860). From this become the cosmid clones
Qc8D 1 1 , Qc3F1 2 und Qc1 2G1 1 (vgl. Kioschis, P. et al., Cytogenet. Cell. Genet. 58, ( 1991 ), 2070; Kioschis, P. et al., Genomics 33, ( 1996), im Druck), verwendet und einer cDNA Selektion (vgl. Korn, B. et al., Mol. Genet. 4 ( 1 992), 235-242), unterworfen, wodurch das cDNA-Fragmeπt 79g1 P5 erhalten wird. Dieses wird zur Hybridisierung einer cDNA-Bibliothek von humaner Placenta (z.B.Qc8D 1 1, Qc3F1 2 and Qc1 2G1 1 (see. Kioschis, P. et al., Cytogenet. Cell. Genet. 58, (1991), 2070; Kioschis, P. et al., Genomics 33, (1996), in print), and subjected to cDNA selection (cf. Korn, B. et al., Mol. Genet. 4 (1 992), 235-242), whereby the cDNA fragment 79g1 P5 is obtained. This is used to hybridize a human placenta cDNA library (e.g.
STRATAGENE, Kat. Nr. 936203) verwendet. Es wird eine erfindungsgemäße cDNA erhalten.STRATAGENE, Cat. No. 936203) was used. A cDNA according to the invention is obtained.
Eine erfindunggemäße cDNA kann in einem Vektor bzw. Expressionsvektor vorliegen. Beispiele solcher sind dem Fachmann bekannt. Im Falle eines Ex¬ pressionsvektors für E. coli sind dies z.B. pGEMEX, pUC-Derivate, pGEX-2T, pET3b und pQE-8, wobei letzterer bevorzugt ist. Für die Expression in Hefe sind z.B. pY100 und Ycpad i zu nennen, während für die Expression in tierischen Zellen z.B. pKCR, pEFBOS, cDM8 und pCEV4, anzugeben sind. Für die Ex¬ pression in insektenzellen eignet sich besonders der Baculovirus-Expressions- vektor pAcSGHisNT-A.A cDNA according to the invention can be present in a vector or expression vector. Examples of such are known to the person skilled in the art. In the case of an expression vector for E. coli, these are, for example, pGEMEX, pUC derivatives, pGEX-2T, pET3b and pQE-8, the latter being preferred. For expression in yeast Examples include pY100 and Ycpad i, while pKCR, pEFBOS, cDM8 and pCEV4 must be specified for expression in animal cells. The baculovirus expression vector pAcSGHisNT-A is particularly suitable for expression in insect cells.
Der Fachmann kennt geeignete Zellen, um eine, erfindungsgemäße, in einem Expressionsvektor vorliegende cDNA zu exprimieren. Beispiele solcher Zellen umfassen die E.coli-Stämme HB101 , DH1 , x1 776, JM101 , JM 109, BL21 und SG 1 3009, wobei letzterer bevorzugt ist, den Hefe-Stamm Saccharomyces cerevisiae und die tierischen Zellen L, 3T3, FM3A, CHO, COS, Vero und HeLa sowie die Insektenzellen sf9.The person skilled in the art knows suitable cells in order to express a cDNA according to the invention which is present in an expression vector. Examples of such cells include the E. coli strains HB101, DH1, x1 776, JM101, JM 109, BL21 and SG 1 3009, the latter being preferred, the yeast strain Saccharomyces cerevisiae and the animal cells L, 3T3, FM3A, CHO , COS, Vero and HeLa and the insect cells sf9.
Der Fachmann weiß, in welcher Weise eine erfindungsgemäße cDNA in einen Expressionsvektor inseriert werden muß. Ihm ist auch bekannt, daß diese DNA in Verbindung mit einer für ein anderes Protein bzw. Peptid kodierenden DNA inseriert werden kann, so daß die erfindungsgemäße cDNA in Form eines Fu¬ sionsproteins exprimiert werden kann.The person skilled in the art knows how a cDNA according to the invention has to be inserted into an expression vector. He is also aware that this DNA can be inserted in conjunction with a DNA coding for another protein or peptide, so that the cDNA according to the invention can be expressed in the form of a fusion protein.
Des weiteren kennt der Fachmann Bedingungen, transformierte bzw. trans- fizierte Zellen zu kultivieren. Auch sind ihm Verfahren bekannt, das durch die erfindungsgemäße cDNA exprimierte Protein zu isolieren und zu reinigen. Ein solches Protein, das auch ein Fusionsprotein sein kann, ist somit ebenfalls Gegenstand der vorliegenden Erfindung.Furthermore, the person skilled in the art knows the conditions for cultivating transformed or transfected cells. He is also aware of methods for isolating and purifying the protein expressed by the cDNA according to the invention. Such a protein, which can also be a fusion protein, is thus also the subject of the present invention.
Ein weiterer Gegenstand der vorliegenden Erfindung ist ein gegen ein vorstehen¬ des Protein bzw. Fusionsprotein gerichteter Antikörper. Ein solcher Antikörper kann durch übliche Verfahren hergestellt werden. Er kann polyklonal bzw. monoklonal sein. Zu seiner Herstellung ist es günstig, Tiere, insbesondere Kaninchen oder Hühner für einen polyklonalen und Mäuse für einen monoklona- len Antikörper, mit einem vorstehenden (Fusions)protein oder Fragmenten davon zu immunisieren. Weitere "Booster" der Tiere können mit dem gleichen (Fu- sionslprotein oder Fragmenten davon erfolgen. Der polyklonale Antikörper kann dann aus dem Serum bzw. Eigelb der Tiere erhalten werden. Für den monoklo- nalen Antikörper werden Milzzellen der Tiere mit Myelomzellen fusioniert.Another object of the present invention is an antibody directed against a protruding protein or fusion protein. Such an antibody can be produced by conventional methods. It can be polyclonal or monoclonal. For its production, it is favorable to immunize animals, in particular rabbits or chickens for a polyclonal and mice for a monoclonal antibody, with an above (fusion) protein or fragments thereof. Further "boosters" of the animals can be carried out with the same (fusion protein or fragments thereof. The polyclonal antibody can then be obtained from the serum or egg yolk of the animals. For the monoclonal antibody, animal spleen cells are fused with myeloma cells.
Die vorliegende Erfindung ermöglicht es, Störungen der Zelldifferenzierung, insbesondere bei Muskelzellen und ganz besonders bei myotubulärer Myopathie, in ihrer Ursache zu untersuchen. Mit einer erfindungsgemäßen Nukleinsäure, insbesondere einer DNA, und hiervon abgeleiteten Primern, kann in Säugetieren, insbesondere dem Menschen festgestellt werden, ob sie ein Gen enthalten und/oder exprimieren, das für ein verkürztes bzw. mutiertes (TVP) in vorstehen- dem Sinne kodiert. Hierzu wird der Fachmann übliche Verfahren, wie ReverseThe present invention makes it possible to investigate the causes of cell differentiation disorders, particularly in muscle cells and very particularly in myotubular myopathy. With a nucleic acid according to the invention, in particular a DNA, and primers derived therefrom, it can be determined in mammals, in particular humans, whether they contain and / or express a gene which codes for a truncated or mutated (TVP) in the above sense . To this end, the person skilled in the art uses customary methods, such as reverse
Transkiption, PCR-Reaktion, Hybridisierung und Sequenzierung, durchführen. Erfindungsgemäß wird auch ein Kit bereitgestellt, der eine vorstehende Nu¬ kleinsäure, insbesondere DNA, und/oder hiervon abgeleitete Primer sowie Träger und übliche Hilfsstoffe enthält.Perform transcription, PCR reaction, hybridization and sequencing. According to the invention, a kit is also provided which contains the above nucleic acid, in particular DNA, and / or primers derived therefrom, as well as carriers and customary auxiliaries.
Ferner eignet sich die vorliegende Erfindung, therapeutische Maßnahmen bei Störungen der Zelldifferenzierung, insbesondere bei Muskelzellen und ganz besonders bei myotubulärer Myopathie, zu ergreifen. Ein erfindungsgemäßes (TVP) kann in Säugetiere, insbesondere den Menschen, eingebracht werden. Hierzu kann es günstig sein, (TVP) an ein vom jeweiligen Körper nicht als fremd angesehenes Protein, z.B. Transferrin oder BSA, zu koppeln. Auch kann eine erfindungsgemäße Nukleinsäure, insbesondere eine DNA, in Säugetiere, ins¬ besondere den Menschen, eingebracht und dort exprimiert werden. Hierzu kann es günstig sein, die Expression der erfindungsgemäßen Nukleinsäure unter die Kontrolle eines Gewebe-spezifischen Promotors, insbesondere eines Muskel¬ spezifischen Promotors, zu stellen. Mit einem erfindungsgemäßen Antikörper kann die Expression von (TVP) kontrolliert und reguliert werden.Furthermore, the present invention is suitable for taking therapeutic measures in the event of disturbances in cell differentiation, in particular in muscle cells and very particularly in myotubular myopathy. An inventive (TVP) can be introduced into mammals, especially humans. For this purpose, it may be beneficial to (TVP) a protein that is not considered foreign by the respective body, e.g. Transferrin or BSA to couple. A nucleic acid according to the invention, in particular a DNA, can also be introduced into mammals, in particular humans, and expressed there. For this purpose, it may be expedient to place the expression of the nucleic acid according to the invention under the control of a tissue-specific promoter, in particular a muscle-specific promoter. The expression of (TVP) can be controlled and regulated with an antibody according to the invention.
Die vorliegende Erfindung stellt somit einen großen Beitrag zur diagnostischen und therapeutischen Erfassung von Störungen der Zelldifferenzierung, insbeson¬ dere bei Muskelzellen und ganz besonders bei myotubulärer Myopathie, dar. Die diagnostische Erfassung kann dabei nicht nur post- sondern bereits auch präna- tal erfolgen.The present invention thus makes a major contribution to the diagnostic and therapeutic detection of disorders of cell differentiation, in particular in muscle cells and very particularly in myotubular myopathy. valley.
Kurze Beschreibung der Zeichnung:Brief description of the drawing:
Fig. 1 zeigt die Basensequenz und die davon abgeleitete Aminosäurese¬ quenz eines erfindungsgemäßen (TVP),1 shows the base sequence and the amino acid sequence derived therefrom of an inventive (TVP),
Die vorliegende Erfindung wird durch die nachstehenden Beispiele erläutert.The present invention is illustrated by the following examples.
Beispiel 1 : Herstellung und Reinigung eines erfindungsgemäßen (TVP)Example 1: Production and cleaning of an inventive (TVP)
Zur Herstellung eines erfindungsgemäßen (TVP) wurde die DNA von Fig. 1 als Template verwendet. Es wurde ein PCR- Verfahren durchgeführt. Als Primer-Paar wurde verwendet:The DNA of FIG. 1 was used as a template to produce an inventive (TVP). A PCR procedure was carried out. The following was used as a primer pair:
MTM-F: 5'-CAGGGATCCGATGGCAGCCGAGCAGCCTGGCAAC-3' und MTM-R: 5'-GGGGGATCCTCAGAAGTGAGTTTGCACATGGGG-3'MTM-F: 5'-CAGGGATCCGATGGCAGCCGAGCAGCCTGGCAAC-3 'and MTM-R: 5'-GGGGGATCCTCAGAAGTGAGTTTGCACATGGGG-3'
Der PCR-Ansatz bzw. die PCR-Bedingungen waren wie folgt:The PCR approach and the PCR conditions were as follows:
PCR-AnsatzPCR approach
Template DNA (Fig. 1 ) 1μ\ = 1 ngTemplate DNA (Fig. 1) 1μ = 1 ng
Pfu-Polymerase 10x-Puffer 10μl = 1 xPfu polymerase 10x buffer 10μl = 1 x
DMSO , 0μ\ = 10 % dNTP's 1/;L = je 200//MDMSO, 0μ \ = 10% dNTP's 1 /; L = 200 // M each
Oligonukleotide, je 1 ,5μl 3μ\ = je 150 ngOligonucleotides, 1, 5μl 3μ \ = 150 ng each
H2O-bidest ad 99//I PCR-BedingungenH 2 O bidest ad 99 // I PCR conditions
- 92°C - 5 min- 92 ° C - 5 min
- Zugabe von ιμ\ Pfu-Polymerase (Stratagene) = 2,5 Einheiten- Add ιμ \ Pfu polymerase (Stratagene) = 2.5 units
- Zugabe von Paraffin- Add paraffin
PCRPCR
92°C 1 min92 ° C 1 min
58°C 1 min 1 Zyklus 72°C 10 min58 ° C 1 min 1 cycle 72 ° C 10 min
92°C 1 min92 ° C 1 min
58°C 1 min 39 Zyklen58 ° C 1 min 39 cycles
72°C 2 min72 ° C 2 min
72°C 10 min 1 Zyklus72 ° C 10 min 1 cycle
Die amplifizierte DNA wurde jeweils mit Bam Hl gespalten und in den mit Barn Hl gespaltenen Expressionsvektors pQE-8 (Diagen) inseriert. Es wurde das Expressionsplasmid pQ/TVP erhalten. Ein solches kodiert für ein Fusionsprotein aus 6 Histidin-Resten (N-Terminuspartner) und dem erfindungsgemäßen (TVP) von Fig. 1 (C-Terminuspartner). pQ/TVP wurde zur Transformation von E.coli SG 13009(vgl. Gottesman, S. et al., J. Bacteriol. 148y (1981 ), 265-273) verwen¬ det. Die Bakterien wurden in einem LB-Medium mit 100//g/ml Ampicillin und 25μg/ml Kanamycin kultiviert und 4 h mit 60μM Isopropyl-ß-D-Thiogalactopyra- nosid (IPTG) induziert. Durch Zugabe von 6 M Guanidinhydrochlorid wurde eineThe amplified DNA was in each case cleaved with Bam HI and inserted into the expression vector pQE-8 (Diagen) cleaved with Barn HI. The expression plasmid pQ / TVP was obtained. Such a code for a fusion protein of 6 histidine residues (N-terminus partner) and the inventive (TVP) of Fig. 1 (C-terminus partner). pQ / TVP was used to transform E.coli SG 13009 (cf. Gottesman, S. et al., J. Bacteriol. 148 y (1981), 265-273). The bacteria were cultured in an LB medium with 100 g / ml ampicillin and 25 μg / ml kanamycin and induced for 4 h with 60 μM isopropyl-β-D-thiogalactopyranoside (IPTG). A 6 M guanidine hydrochloride was added
Lyse der Bakterien erreicht, anschließend wurde mit dem Lysat eine Chromato¬ graphie (Ni-NTA-Resin) in Gegenwart von 8 M Harnstoff entsprechend der Angaben des Herstellers (Diagen) des Chromatographie-Materials durchgeführt. Das gebundene Fusionsprotein wurde in einem Puffer mit pH 3,5 eluiert. Nach seiner Neutralisierung wurde das Fusionsprotein einer 18 % SDS-Polyacrylamid-Lysis of the bacteria was achieved, then a chromatography (Ni-NTA resin) was carried out with the lysate in the presence of 8 M urea in accordance with the manufacturer's (Diagen) instructions for the chromatography material. The bound fusion protein was eluted in a pH 3.5 buffer. After neutralization, the fusion protein was an 18% SDS polyacrylamide
Gelelektrophorese unterworfen und mit Coomassie-Blau angefärbt (vgl. Thomas, J.O. und Kornberg, R.D., J.Mol. Biol. 149 (1975), 709-733). Es zeigte sich, daß ein erfindungsgemäßes (Fusions)protein in hochreiner Form hergestellt werden kann.Subject to gel electrophoresis and stained with Coomassie blue (cf. Thomas, JO and Kornberg, RD, J. Mol. Biol. 149 (1975), 709-733). It has been shown that a (fusion) protein according to the invention can be produced in highly pure form.
Beispiel 2: Herstellung und Nachweis eines erfindungsgemäßen AntikörpersExample 2: Production and detection of an antibody according to the invention
Ein erfindungsgemäßes Fusionsprotein von Beispiel 1 wurde einer 1 8 % SDS- Polyacrylamid-Gelelektrophorese unterzogen. Nach Anfärbung des Gels mit 4 M Natriumacetat wurde eine ca. 205 kD Bande aus dem Gel herausgeschnitten und in Phosphat gepufferter Kochsalzlösung inkubiert. Gel-Stücke wurden sedimentiert, bevor die Proteinkonzentration des Überstandes durch eine SDS- Polyacrylamid-Gelelektrophorese, der eine Coomassie-Blau-Färbung folgte, bestimmt wurde. Mit dem Gel-gereinigten Fusionsprotein wurden Tiere wie folgt immunisiert:A fusion protein according to the invention from Example 1 was subjected to 1 8% SDS-polyacrylamide gel electrophoresis. After staining the gel with 4 M sodium acetate, an approximately 205 kD band was cut out of the gel and incubated in phosphate-buffered saline. Pieces of gel were sedimented before the protein concentration of the supernatant was determined by SDS-polyacrylamide gel electrophoresis followed by a Coomassie blue stain. Animals were immunized with the gel-purified fusion protein as follows:
Immunisierungsprotokoll für polyklonale Antikörper im KaninchenImmunization protocol for polyclonal antibodies in rabbits
Pro Immunisierung wurden 35μg Gel-gereinigtes Fusionsprotein in 0,7 ml PBS und 0,7 ml komplettem bzw. inkomplettem Freund's Adjuvans eingesetzt.35 μg of gel-purified fusion protein in 0.7 ml of PBS and 0.7 ml of complete or incomplete Freund's adjuvant were used for each immunization.
Tag O: 1 . Immunisierung (komplettes Freund's Adjuvans)Day O: 1st Immunization (Freund's Complete Adjuvant)
Tag 14 2. Immunisierung (inkomplettes Freund's Adjuvans; icFA) Tag 28 3. Immunisierung (icFA) Tag 56 4. Immunisierung (icFA) Tag 80 AusblutenDay 14 2nd immunization (incomplete Freund's adjuvant; icFA) Day 28 3rd immunization (icFA) Day 56 4th immunization (icFA) Day 80 Bleeding
Das Serum des Kaninchens wurde im Immuπoblot getestet. Hierzu wurde ein erfindungsgemäßes Fusionsprotein von Beispiel 1 einer SDS-Polyacrylamid- Gelelektrophorese unterzogen und auf ein Nitrocellulosefilter übertragen (vgl. Khyse-Andersen, J., J. Biochem. Biophys. Meth. 10, ( 1 984), 203-209). DieThe rabbit serum was tested in the immunoblot. For this purpose, a fusion protein according to the invention from Example 1 was subjected to SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose filter (cf. Khyse-Andersen, J., J. Biochem. Biophys. Meth. 10, (1 984), 203-209). The
Western Blot-Analyse wurde wie in Bock, C.-T. et al. , Virus Genes 8, (1 994), 21 5-229, beschrieben, durchgeführt. Hierzu wurde das Nitrocellulosefilter eine Stunde bei 37°C mit einem ersten Antikörper inkubiert. Dieser Antikörper war das Serum des Kaninchens ( 1 : 10000 in PBS). Nach mehreren Waschschritteπ mit PBS wurde das Nitrocellulosefilter mit einem zweiten Antikörper inkubiert. Dieser Antikörper war ein mit alkalischer Phosphatase gekoppelter monoklonaler Ziege Anti-Kaninchen-lgG-Antikörper (Dianova) (1 :5000) in PBS. Nach 30- minütiger Inkubation bei 37°C folgten mehrere Waschschritte mit PBS und anschließend die alkalische Phosphatase-Nachweisreaktion mit Entwicklerlösung (36/yM 5' Bromo-4-chloro-3-indolylphosphat, 400μM Nitroblau-tetrazolium, 100mM Tris-HCI, pH 9.5, 100 mM NaCI, 5 mM MgCI2) bei Raumtemperatur, bis Banden sichtbar waren.Western blot analysis was performed as in Bock, C.-T. et al. , Virus Genes 8, (1 994), 21 5-229. For this purpose, the nitrocellulose filter became a Incubated for one hour at 37 ° C with a first antibody. This antibody was rabbit serum (1: 10,000 in PBS). After several washing steps with PBS, the nitrocellulose filter was incubated with a second antibody. This antibody was an alkaline phosphatase-coupled goat anti-rabbit IgG monoclonal antibody (Dianova) (1: 5000) in PBS. After 30 minutes incubation at 37 ° C, several washing steps with PBS followed and then the alkaline phosphatase detection reaction with developer solution (36 / y M 5 'bromo-4-chloro-3-indolylphosphate, 400μM nitroblue tetrazolium, 100mM Tris-HCl, pH 9.5, 100 mM NaCl, 5 mM MgCl 2 ) at room temperature until bands were visible.
Es zeigte sich, daß erfindungsgemäße, polyklonale Antikörper hergestellt werden können.It was found that polyclonal antibodies according to the invention can be produced.
Immunisierungsprotokoll für polyklonale Antikörper im HuhnImmunization protocol for chicken polyclonal antibodies
Pro Immunisierung wurden 40μg Gel-gereinigtes Fusionsprotein in 0,8 ml PBS und 0,8 ml komplettem bzw. inkomplettem Freund's Adjuvans eingesetzt.For each immunization, 40 μg gel-purified fusion protein in 0.8 ml PBS and 0.8 ml complete or incomplete Freund's adjuvant were used.
Tag O. 1 . Immunisierung (komplettes Freund's Adjuvans)Day O.1 Immunization (Freund's Complete Adjuvant)
Tag 28: 2. Immunisierung (inkomplettes Freund's Adjuvans; icFA)Day 28: 2nd immunization (incomplete Freund's adjuvant; icFA)
Tag 50: 3. Immunisierung (icFA)Day 50: 3rd immunization (icFA)
Aus Eigelb wurden Antikörper extrahiert und im Western Blot getestet. Es wurden erfindungsgemäße, polyklonale Antikörper nachgewiesen.Antibodies were extracted from egg yolk and tested in a Western blot. Polyclonal antibodies according to the invention have been detected.
Immunisierungsprotokoll für monoklonale Antikörper der MausImmunization protocol for mouse monoclonal antibodies
Pro Immunisierung wurden 12μg Gel-gereinigtes Fusionsprotein in 0,25 ml PBS und 0,25 ml komplettem bzw. inkomplettem Freund's Adjuvans eingesetzt; bei der 4. Immunisierung war das Fusionsprotein in 0,5 ml (ohne Adjuvans) gelöst. Tag O. 1. Immunisierung (komplettes Freund's Adjuvans) Tag 28 2. Immunisierung (inkomplettes Freund's Adjuvans; icFA) Tag 56 3. Immunisierung (icFA) Tag 84 4. Immunisierung (PBS) Tag 87 FusionFor each immunization, 12 μg gel-purified fusion protein in 0.25 ml PBS and 0.25 ml complete or incomplete Freund's adjuvant were used; at the 4th immunization the fusion protein was dissolved in 0.5 ml (without adjuvant). Day O. 1. Immunization (Freund's complete adjuvant) Day 28 2. Immunization (Freund's incomplete adjuvant; icFA) Day 56 3. Immunization (icFA) Day 84 4. Immunization (PBS) Day 87 Fusion
Überstände von Hybridomen wurden im Western Blot getestet. Erfindungs¬ gemäße, monoklonale Antikörper wurden nachgewiesen. Supernatants from hybridomas were tested in a Western blot. Monoclonal antibodies according to the invention have been detected.

Claims

Patentansprüche claims
1 . Tyrosin-Phosphatase-verwandtes Protein, wobei das Protein die Amino¬ säuresequenz von Fig. 1 oder eine hiervon durch eine oder mehrere Aminsosäuren unterschiedliche Aminosäuresequenz umfaßt.1 . Tyrosine phosphatase-related protein, the protein comprising the amino acid sequence of FIG. 1 or an amino acid sequence different therefrom by one or more amino acids.
2. DNA, kodierend für das Protein nach Anspruch 1 , wobei die DNA umfaßt: (a) die DNA von Fig. 1 oder eine hiervon durch ein oder mehrere Ba¬ senpaare unterschiedliche DNA, (b) eine mit der DNA von (a) hybridisierende DNA oder2. DNA coding for the protein according to claim 1, the DNA comprising: (a) the DNA of FIG. 1 or a DNA different therefrom by one or more base pairs, (b) one with the DNA of (a) hybridizing DNA or
(c) eine mit der DNA von (a) oder (b) über den degenerierten geneti¬ schen Code verwandte DNA.(c) a DNA related to the DNA of (a) or (b) via the degenerate genetic code.
3. Expressionsplasmid, umfassend die DNA nach Anspruch 2.3. Expression plasmid comprising the DNA of claim 2.
4. Transformante, enthaltend das Expressionsplasmid nach Anspruch 3.4. Transformant containing the expression plasmid according to claim 3.
5. Verfahren zur Herstellung des Proteins nach Anspruch 1 , umfassend die Kultivierung der Transformante nach Anspruch 4 unter geeigneten Bedin- gungen.5. A method for producing the protein according to claim 1, comprising culturing the transformant according to claim 4 under suitable conditions.
6. Antikörper, gerichtet gegen das Protein nach Anspruch 1 .6. Antibody directed against the protein according to claim 1.
7. Verwendung des Proteins nach Anspruch 1 als Reagens zur Therapie einer gestörten Zelldifferenzierung.7. Use of the protein according to claim 1 as a reagent for the therapy of a disturbed cell differentiation.
8. Verwendung nach Anspruch 7, wobei die gestörte Zelldifferenzierung bei myotubulärer Myopathie vorliegt.8. Use according to claim 7, wherein the disturbed cell differentiation is present in myotubular myopathy.
9. Verwendung der DNA nach Anspruch 2 als Reagens zur Diagnose und/- oder Therapie einer gestörten Zelldifferenzierung. 9. Use of the DNA according to claim 2 as a reagent for diagnosis and / or therapy of a disturbed cell differentiation.
10. Verwendung nach Anspruch 9, wobei die gestörte Zelldifferenzierung bei myotubulärer Myopathie vorliegt. 10. Use according to claim 9, wherein the disturbed cell differentiation is present in myotubular myopathy.
EP97918050A 1996-03-21 1997-03-21 Tyrosine-phosphatase-related protein Withdrawn EP0904384A2 (en)

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US8790707B2 (en) 2008-12-11 2014-07-29 3M Innovative Properties Company Surface-treated calcium phosphate particles suitable for oral care and dental compositions
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