Drug – bio-affecting and body treating compositions – Enzyme or coenzyme containing – Transferases
Reexamination Certificate
1999-03-02
2001-11-06
Achutamurthy, Ponnathapu (Department: 1652)
Drug, bio-affecting and body treating compositions
Enzyme or coenzyme containing
Transferases
C435S196000, C435S325000, C435S252300, C435S320100, C536S023200, C514S012200, C514S04400A
Reexamination Certificate
active
06312688
ABSTRACT:
The present invention relates to a tyrosine-phosphatase-related protein, a DNA which codes for such a protein and a process for producing such a protein. In addition, the invention concerns the use of the DNA and protein and antibodies directed against the protein.
Tyrosine kinase and tyrosine phosphatase are enzymes which have opposite effects. Tyrosine kinase effects the phosphorylation of certain tyrosine residues in proteins, whereas tyrosine phosphatase reverses this phosphorylation again. Both enzymes play an important part for the signal transduction, the control of cell growth and cell differentiation.
Diseases are known which are based on disturbances occurring in Tell differentiation. One of these diseases is myotubular myopathy. It is an X-chromosomal-connected disease which is accompanied by altered muscle cells. This disease manifests itself in general muscle weakness, particularly spontaneous movements are hardly possible. Likewise, the respiration of newborns is strongly confined. This frequently leads to premature death However, the causes of disturbed cell differentiation in the case of myotubular myopathy are not known.
Therefore, it is the object of the present invention to provide a product by which it is possible to investigate the cause of cell differentiation disturbances, particularly in the case of myotubular myopathy, and optionally treat them. According to the invention this is achieved by the subject matters defined in the claims.
Thus, the subject matter of the present invention relates to a tyrosine-phosphatase-related protein, the protein comprising the amino acid sequence of
FIG. 1
or an amino acid sequence differing therefrom by one or several amino acids.
The present invention is based on the applicant's finding that a protein exists in animals, particularly mammals, more particularly human beings, which has homologies with known tyrosine phosphatases and also tyrosine phosphatase activity but differs from known tyrosine phosphatases on the DNA level by hybridization under normal conditions. Such a protein has the amino acid sequence of
FIG. 1
or an amino acid sequence differing therefrom by one or several amino acids. The applicant has also discovered that the protein is important for cell differentiation. He has found that, in its shortened form and mutated form, respectively, i.e. without or only with restricted tyrosine phosphatase activity, this protein disturbs the differentiation of cells, particularly muscle cells, and more particularly leads to the formation of myotubular myopathy.
The above protein is referred to as “tyrosine-related protein” (TVP) in the present invention.
A further subject matter of the present invention relates to a nucleic acid coding for (TVP). It may be an RNA or a DNA. The latter may be e.g. a genomic DNA or a cDNA. Preferred is a DNA comprising the following:
(a) the DNA of
FIG. 1
or a DNA differing therefrom by one or several base pairs,
(b) a DNA hybridizing with the DNA of (a), or
(c) a DNA related to the DNA of (a) or (b) via the degenerated genetic code.
The expression “hybridizing DNA” refers to a DNA which hybridizes with a DNA of (a) under normal conditions, particularly at 20° C. below the melting point of the DNA.
The DNA of
FIG. 1
was deposited wit h the DSM (Deutsche Sammlung von Mikroorganismen und Zellkulturen [German-type collection of micro-organisms and cell cultures]) as hp6 under DSM 10558 on Mar. 4, 1996.
A DNA according to the invention is described below in the form of a cDNA. It is exemplary for every DNA falling under the present invention.
For preparing a cDNA according to the invention, it is favorable to use as a basis 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, (1991), 2070) which was prepared from the cell hybrid QIZ (cf. Warren, S. T. et al., Proc. Natl. Acad. Scif U.S.A. 87 (1990), 3856-3860). The cosmid clones Qc8D11, Qc3F12 and Qc12G11 thereof (cf. Kioschis, P. et al., Cytogenet. Cell. Genet. 58, (1991), 2070; Kioschis, P. et al., Genomics 33, (1996) in print) are used and subjected to DNA selection (cf. Korn, B. et al., Mol. Genet. 4 (1992), 235-242) s o as to obtain the cDNA fragment 79g1P5. It is used for hybridizing a cDNA library of human placenta (e.g. STRATAGENE, catalog No. 936203). A cDNA according to the invention is obtained.
A cDNA according to the invention may be present in a vector and expression vector, respectively. A person skilled in the art is familiar with examples thereof. In the case of an expression vector for
E. coli
these are e.g. pGEMEX, pUC derivatives, pGEX-2T, pET3b and pQE-8, the latter being preferred. For the expression in yeast, e.g. pY100 and Ycpad1 have to be mentioned while e.g. pkCR, pEFBOS, cDM8 and pCEV4 have to be indicated for the expression in animal cells . The baculovirus expression vector pAcSGHisNT-A is especially suitable for the expression in insect cells.
The person skilled in the art is familiar with suitable cells to express a cDNA according to the invention, which is present in an expression vector. Examples of such cells comprise the
E. coli
strains RB101, DH1, x1776, JM101, JM109, BL21 and SG13009, the latter being preferred, the yeast strain saccharomyces cerevisiae and the animal cells L, 3T3, FM3A, CHO, COS, Vero and HeLa as well as the insect cells sf9.
The person skilled in the art knows in which way a DNA according to the invention has to be inserted in an expression vector. He is also familiar with the fact that this DNA can be inserted in combination with a DNA coding for another protein and peptide, respectively, so that the cDNA according to the invention can be expressed in the form of a fusion protein.
In addition, the person skilled in the art knows conditions of culturing transformed cells and transfected cells, respectively. He is also familiar with processes of isolating and purifying the protein expressed by the cDNA according to the invention. Thus, such a protein, which may also be a fusion protein, is also a subject matter of the present invention.
A further subject matter of the present invention relates to an antibody directed against an above protein and fusion protein, respectively. Such an antibody can be prepared by common methods. It may be polyclonal and monoclonal, respectively. For its preparation it is favorable to immunize animals—particularly rabbits or chickens for a polyclonal antibody and mice for a monoclonal antibody—with an above (fusion) protein or with fragments thereof. Further “boosters” of the animals can be effected with the same (fusion) protein or with fragments thereof. The polyclonal antibody may then be obtained from the animal serum and egg yolk, respectively. For the preparation of the monoclonal antibody, animal spleen cells are fused with myeloma cells.
The present invention enables to investigate the causes of cell differentiation disturbances, particularly in the case of muscle cells, and more particularly in the case of myotubular myopathy. By means of a nucleic acid according to the invention, particularly a DNA, and primers derived therefrom, it can be determined in mammals, particularly human beings, whether they contain and/or express a gene which codes, within the above sense, for a shortened (TVP) and mutated (TVP), respectively. For this purpose, a person skilled in the art will carry out common methods such as reverse transcription, PCR reaction, hybridization and sequencing. A kit which contains an above nucleic acid, particularly DNA, and/or primers derived therefrom as well as carriers and conventional auxiliary agents, is also provided according to the invention.
Furthermore, the present invention is suited to take therapeutic measures in the case of cell differentiation disturbances, particularly in the case of muscle cells and more particularly in the case of myotubular myopathy. A (TVP) according to the invention can be inserted in mammals, particularly human beings. For this purpose,
Dahl Niklas
Hu Ling Jia
Kioschis Petra
Laporte Jocelyn
Mandel Jean Louis
Achutamurthy Ponnathapu
Burns Doane Swecker & Mathis L.L.P.
Deutsches Krebsotorschungszentrum Stiftung des Offentlichen Rech
Monshipouri Maryam
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