EP0652959A1

EP0652959A1 - Isolation and cloning of a protein with tyrosine-phosphatase activity

Info

Publication number: EP0652959A1
Application number: EP93917704A
Authority: EP
Inventors: Lucia Monaco; Antonella Isacchi
Original assignee: Pharmacia SpA
Current assignee: Pfizer Italia SRL
Priority date: 1992-07-31
Filing date: 1993-07-29
Publication date: 1995-05-17
Also published as: MX9304580A; WO1994003610A3; ZA935483B; IL106505A0; WO1994003610A2; AU4704793A; CN1082110A; GB9216328D0

Abstract

Mammalian receptor-type protein tyrosine phosphatases (PTPases) whose extracellular domains include an Arg-Gly-Asp sequence, and functional derivatives thereof. Examples of such PTPases are novel members of the Class II PTPase group.

Description

ISOLATION AND CLONING OF A PROTEIN WITH

TYROSINE-PHOSPHATASE ACTIVITY

Field of the Invention The present invention relates to transmembrane tyrosine phosphatase proteins, DNA coding therefor, methods for production and identification of these proteins, and methods for screening compounds capable of binding to and inhibiting or stimulating phosphatase enzymatic activity.

Background of the Invention

The phosphorylation state of protein tyrosyl residues plays a key role in cellular growth control. Cellular phosphotyrosine levels are regulated by the relative activities of opposing protein-tyrosine kinases and tyrosine phosphatases. Several growth factor receptors, as well as insulin receptor, are tyrosine-specific protein kinases.

The activation of receptor tyrosine kinases is mediated by interaction with their respective growth factors or cytokines, such as FGF, EGF, PDGF, GM-CSF and IL-1, and usually induces mitogenesis and growth (Yarden, Y. and Ullrich, A.: Annual Rev. Biochem. 52, 443-478; 1988). The association between tyrosine phosphorylation and cell proliferation was further supported by the finding that many όncogene products also act as tyrosine kinases and share extensive sequence homology with known polypeptide growth factor receptors. These observations suggest that protein tyrosine phosphatases, for their ability to selectively counteract the action of tyrosine kinases, may play an important role in the control of cell growth and activation. Accordingly, it has been demonstrated that inhibitors of tyrosine phosphatases can "reversibly transform" cells and it has been proposed that alterations in PTPase activity may be involved in neoplastic processes, leading to the concept that protein tyrosine phosphatases may act as tumor suppressors. (Klarlund, J.K. (1985) Cell 41, 707-717).

Although much of the work on tyrosine kinases has focused on growth regulation, recent findings in the non- proliferating cells of the central nervous system demonstrate the presence of relatively large amounts of tyrosine kinase activity. For example, receptors for insulin are present in specific brain regions and are specifically autophosphorylated following ligand stimulation. It has recently been demonstrated that the distribution of the insulin receptor in rat brain is strictly correlated with the distribution of phosphotyrosines, suggesting that this kinase is active in vivo and participates to the functions of adult central nervous system (Moss, A.M., Unger, J.W., Moxley, R.T. and Livingston, J. Proc. Natl. Acad. Sci. l_ 4453-4457, 1990). On the other hand, several tyrosine phosphatases have recently been reported to be selectively expressed in central nervous system, suggesting that they may be involved in regulation of the tyrosine kinase activation in brain.

If these enzymes are involved in controlling tyrosine phosphorylation pathways, their activity, like that of tyrosine kinases, should be modulated in response to specific cellular events or ligand interactions. Indeed, some of the phosphatases that have been isolated so far have shown a putative receptor- type structure, suggesting that their activity is regulated by an external ligand in a finely regulated cascade of events. It is clear that the isolation of a tyrosine phosphorylation ligand could represent an important step towards the inhibition of uncontrolled cell growth such as in the case of malignancies. On the basis of their structural organization, protein tyrosine phosphatase (PTPases) can be divided into three classes. Class I contains the low molecular weight non-receptor molecules possessing a single catalytic domain and includes placental PTPase IB (Charbonneau, H. et al., Proc. Natl. Acad. Sci. 86; 5252-5256, 1989; Chernoff, J. et al., Proc. Natl. Acad. Sci. 82:2735-2789,1990), T-cell PTPase (Cool, D.E. et al., Proc. Natl. Acad. Sci. j$6_:5257-5261,1989) and rat brain PTPase (Guan,K. et al., Proc. Natl, Acad. Sci. £2:1501-1505,1990). Class II and III PTPases are receptor-like transmembrane receptors. While the sole members of Class II described so far (HPTP3 and DPTP₁₀D) have a single cytoplasmic catalytic domain (Kreuger, N.X. et al.: EMBO J., 9, 3241-3252, 1990; Shin-Shay, T. et al : Cell 67, 675-685, 1991) , Class III members possess two repeated putative catalytic domains in the cytoplasmic region of the molecule. Class III includes the leukocyte common antigen (LCA) (Ralph, S.J., EMBO J., .6:1251-1257, 1987; Charbonneau, H. et al., Proc. Natl. Acad. Sci. 8.5:7182-7186, 1988), the LCA-related protein, LAR (Streuli, M. et al., J. Exp. Med., 168. - 1523-1530, 1988), the LAR-related Drosophila proteins DLAR and DPTP (Streuli, M. et al., Proc. Natl. Acad. Sci. J36:8698-8702, 1989) and the human (Krueger, N.X., Streuli, M. and Saito, H. , EMBO J. 9_, 3241-3252, 1990; Kaplan, R. et al., Proc. Natl. Acad. Sci., 82, 7000-7004, 1990) and mouse (Sap, J., et al.: Proc. Natl. Acad. Sci., 82, 6112- 6116, 1990; Mathews, R. et al.: Proc. Natl. Acad., Sci, 2, 4444- 4448, 1990) proteins HPTP α, HPTP γ, HPTP <S, HPTP €.

There is significant amino acid homology and evolutionary conservation both between the catalytic domains of different phosphatases as well as between repeated domains 1 and 2 within the same PTPase. In particular, the amino acid sequence VHCSXG (one letter code, X is preferentially A or D) , seems to be part of the active site of the enzyme (Streuli, M. Krueger, N.X., Thai, T., Tang, M. and Saito, H. EMBO J. 9_, 2399-2407, 1990).

Several PTPases have adhesion molecule-like extracellular domains consisting of Fibronectin-type (FN) repeats alone (HPTP0) or of immunoglobulin domains and FN repeats together (LAR, DLAR and DPTP) , suggesting that they may couple cell-cell recognition to tyrosine phosphorylation. Indeed, it has been shown that density-dependent inhibition of cell growth involves the regulated elevation of tyrosine phosphatase activity, thus supporting the concept that this activity may play a role in the control of cell growth, differentiation and oncogenesis.

Summary of the invention

In view of the role of tyrosine phosphatases in cell control mechanisms, both as potential anti-oncogenes as well as effectors in newly discovered mechanisms of transmembrane signalling, we have undertaken a search for additional phosphatases potentially involved in such processes.

Accordingly, the invention provides mammalian receptor- type protein tyrosine phosphatases (PTPases) whose extracellular domains include an Arg-Gly-Asp sequence, and functional derivatives thereof. Examples of such PTPases are Class II PTPases. A Class II PTPase may be the novel PTPase PTP35 which has a transmembrane topology. Two different forms of PTP35 have been isolated, sharing common structural characteristics and differing only in their N-terminal sequences. Importantly, the extracellular domain of PTP35 is unrelated to any other receptor-type phosphatase described so far and contains an Arg-Gly-Asp (RGD) sequence. The expression of PTP35 seems to be particularly significant in brain, suggesting that it may have an important role in the control of central nervous system metabolism and differentiation. Moreover, when PTP35 is studied in cultured fibroblasts, it appears to be involved in the control of cell proliferation. Also, the levels of mRNA for phosphatase PTP35 seem to be specifically increased by stimulation of 3T3 cells in culture with basic Fibroblast Growth Factor (bFGF) or other cell mitogens.

Detailed description of the invention

Through the use of recombinant DNA methods, the present inventors have identified a novel receptor-type protein tyrosine phosphatase. The analysis performed at the DNA level as well as the predicted amino acid structure of the proteins indicate that the proteins, lacking duplication of the catalytic domain, belong to Class II of the Protein Tyrosine Phosphatases (PTPases) , thus representing new examples of this particular group of phosphatases.

The different forms of PTP35 described herein share common features. On the basis of the predicted amino acid sequence various functional domains can be recognised. Based on the presence of a hydrophobic stretch (see Figures la and lb) , PTP35 is classified as a transmembrane protein, comprising an extracellular domain N-terminal to the hydrophobic stretch, and an intracellular domain C-terminal to the hydrophobic stretch. The predicted intracellular portion of the protein, which is identical in the different forms of PTP35 so far isolated, consists of a unique domain displaying significant homology to the intracellular catalytic regions of the previously described transmembrane phosphatases. The conserved amino acid sequence IIVHCSDGAGRTG (one letter code) , which has been proposed to be part of the phosphatase catalytic domain, is also present in PTP35 (see Figs, la and lb) . The greatest degree of homology was found with mouse LRPA (Matthews, R.J., Cahir, E.D. and Thomas, M.L. Proc. Natl. Acad. Sci., 82, 4444-4448, 1990; Sap. J. et al.,: Proc. Natl. Acad. Sci, l_, 6112-6116, 1990) and mouse CD45 (Charbonneau, H., Tonks, N. , Walsh, K. and Fischer, E. Proc. Natl. Acad. Sci. JL5, 7182-7186, 1988) tyrosine phosphatases.

In contrast, no significant homology with known extracellular domain sequences was evidenced by the Swissprot database. Perhaps the most interesting feature of the extracellular domain is the presence of an Arg-Gly-Asp (RGD) sequence, which is shared by a number of proteins involved in cell recognition and adhesion mechanisms, including fibronectin,vitronectin, fibrinogen, Von Willebrand factor and protein GPIIbllla. The RGD sequence of each of these proteins is recognized by at least one member of a family of structurally related receptors named integrins which are responsible for cell anchorage related processes. PTP35 is the first tyrosine phosphatase protein found to have an RGD sequence, which may have important biological implications. Other relevant characteristics of the extracellular domain of PTP35 include the presence of 2 potential N-linked glycosylation sites.

The present invention thus includes a new mammalian receptor-type protein tyrosine phosphatase PTPase, named PTP35, whose extracellular domain includes an Arg-Gly-Asp sequence, or any functional derivative thereof. PTP35 does not share any significant homology with other known PTPases in its extracellular domain. The sequences of two forms of PTP35 are shown in Figures la and lb.

The protein of the invention may be of natural origin or, alternatively, may be prepared by chemical or recombinant means. When the molecule is of natural origin it may be obtained by subjecting the cells, tissues or fluids containing the PTPase to standard protein purification techniques so as to obtain a preparation substantially free of other proteins with which the PTPase is natively associated.

An example of purification techniques is represented by an affinity purification in which a solid-phase substrate or ligand binds the enzymatic or, respectively, the receptor domain of the PTPase. Alternatively, the purification can be achieved by a combination of standard methods, such as ammonium sulfate precipitation, molecular sieve chromatography and ion exchange chromatography. The PTPase of the invention may be biochemically purified from a variety of tissues and cell lines. Among these, mouse brain and NIH-3T3 cell line are preferred.

The invention includes a fusion protein comprising a PTPase or derivative thereof according to the invention, fused to a carrier polypeptide.

As already said the protein produced by the above methods is substantially free from other proteins. "Substantially free of other proteins" indicates that the protein is at least 90% or even at least 95% free of other proteins. Most preferably, the protein is 98% of more pure. The term "other proteins" includes proteins with which the PTPase is associated naturally in a cell and proteins present in a recombinant host cell. By "functional derivative" is meant any fragment, variant, analog or chemical derivative of the PTPase, for example of PTP35, retaining at least a portion of the function of the PTPase such as the phosphatase enzymatic activity or the binding of the extracellular domain to a ligand. Suitably, a functional derivative consists essentially of either the intracellular or extracellular domain, or the amino acid sequence encoded by nucleotides 279 to 2874 of Figure la.

By "fragment" it is meant any subset of the molecule, that is, a shorter peptide. By "variant" it is meant a molecule substantially similar to either the entire peptide or a fragment thereof. Natural variants include the homologs of PTP35 in different mammalian species other than mouse, that is the source from which PTP35 has been isolated:' Synthetic variants of a PTPase such as PTP35 may be obtained either by direct chemical synthesis using methods well-known in the art or inserting appropriate mutations in the DNA encoding the PTPase. The variants thus obtained include, for example, deletions, insertions or substitutions of residues as well as extensions. A variant is suitably at least 50%, at least 70%, at least 90%, at least 95% or at least 98% homologous to a PTPase such as PTP35 or fragment thereof. A deletion, insertion, extension or substitution may be N-terminal, C-terminal or internal and comprises one or more amino acids, suitably one, two, three, five, ten, twenty, thirty or forty amino acids. An extension may be much larger and may constitute a carrier protein. A substitution is generally a conservative substitution in which each substituted amino acid is replaced by a biologically similar amino acid. For example, each amino acid may be replaced by an amino acid of similar size, hydrophobicity, charge and/or chemical functionality. Candidate substitutions are:

A for G and vice versa, V by A, L or G;

K by R;

S by T and vice versa; E for D and vice versa; and Q by N and vice versa. By "analog" it is meant a non-natural molecule substantially similar to either the entire molecule or a fragment thereof.

A "chemical derivative" of a PTPase such as PTP35 contains additional chemical moieties not normally a part of the peptide. Examples of chemical modifications include covalent modifications of the peptide, derivatization with bifunctional agents and amidation of the C-terminal carboxyl groups. Such derivatized moieties may improve the physico-chemical characteristics or the biological activity of the PTPase. A particularly preferred embodiment of the present invention is a protein having substantially the amino acid sequence shown in Figure la or lb.

Another embodiment of the present invention is a DNA molecule consisting essentially of a nucleotide sequence encoding a PTPase or functional derivative thereof. The DNA sequence may be in the form of cDNA or genomic DNA. The sequence is suitably that of PTP35 or a derivative thereof.

The recombinant DNA molecules of the present invention can be produced through any of a variety of means well known to the expert in the art and disclosed by, for example, Maniatis et al.. Molecular cloning: a laboratory manual. Second Edition, Cold

Spring Harbor Press, Cold Spring Harbor, NY (1989) .

Single stranded, fully degenerated oligonucleotide probes complementary to a conserved region of the PTPases family are useful for screening DNA, cDNA or RNA preparations derived from cell lines capable of expressing the PTPase gene. A preferred probe would be one directed to the nucleic acid sequence enconding at least 5 amino acid residues of the PTPase of the present invention. Following this or similar techniques the cloning of various genes has been achieved, for example human aldehyde dehydrogenases (Hm, L.C., et al., Proc. Natl. Acad. Sci.

USA 82:3771-3775 (1985)), fibronectin (Suzuki, S. et al. EMBO J.

4_:2519-2524 (1985)), tissue-type plasminogen activator (Pennico, D. et al.. Nature 3.01:214-221 (1983)).

Such methods of cloning can be used even with very small amounts of DNA obtained from an individual, following use of selective amplification techniques. Recombinant DNA methodologies capable of amplifying purified nucleic acid fragments have long been recognized.

Typically, such methodologies involve the introduction of the nucleic acid fragment into a DNA or RNA vector, the clonal amplification of the vector, and the recovery of the amplified nucleic acid fragment. Examples of such methodologies are provided by Cohen et al. (U.S. Patent 4,237,224), Maniatis et al. (Molecular Cloninσ: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989)) which references are herein incorporated by reference. Recently, an in vitro enzymatic method has been described which is capable of increasing the concentration of such desired nucleic acid molecules. This method has been referred to as the "polymerase chain reaction or "PCR" (Mullis, K. et al.. Cold Spring Harbor Symp. Quant. Biol. 5^:263-273 (1986) ; Erlich, H. et alj., EP 50,424; EP 84,796, EP 258,017, EP 237,362; Mullis, K. , EP 201,184; Mullis, K et al.. US 4,683,202; Erlich, H, US 4,582,788; and Saiki, R. et al.. US 4,683,194).

The polymerase chain reaction provides a method for selectively increasing the concentration of a particular nucleic acid sequence even when that sequence has not been previously purified and is present only in a single copy in a particular sample. The method can be used to amplify either single- or double-stranded DNA. The essence of the method involves the use of two oligonucleotide probes to serve as primers for the template-dependent, polymerase mediated replication of a desired nucleic acid molecule.

In a further embodiment, the invention provides a DNA molecule consisting essentially of a nucleotide sequence enconding a functional derivative of PTP35 as defined above. A DNA molecule may encode a variant of a PTPase of the invention. Such a DNA molecule is suitably at least 40%, at least 60%, at least 80%, at least 90%, at least 95% or at least 98% homologous to the sequence showin in Figure 1 or a fragment thereof. A DNA molecule may encode a PTPase of the invention having a deletion, insertion, extension or substitution as described above.

Such a DNA molecule may be prepared by site-directed mutagenesis (as exemplified by Adelman et al.. DNA 2:183 (1983)) of nucleotides in the DNA encoding the polypeptide molecule, thereby producing DNA encoding the derivative, and thereafter expressing the DNA in recombinant cell culture.

In order to express either a native PTPase of the invention or its functional derivatives, the respective DNA coding sequences must be cloned in an appropriate expression vector. An expression vector is a vector which (due to the presence of transcriptional and translational control sequences) is capable of expressing a DNA molecule which has been cloned into the vector and of thereby producing a polypeptide. A vector may be a plasmid or viral vector. Expression of the cloned sequences occurs when the expression vector is introduced into an appropriate host cell. If a prokaryotic expression vector is employed, then the appropriate host cell would be any prokaryotic cell capable of expressing the cloned sequences, for example E. coli. Similarly, if a eukaryotic expression vector is employed, then the appropriate host cell would be any eukaryotic cell capable of expressing the cloned sequences. A yeast host may be employed, for example S. cerevisiae. Alternatively, insect cells may be used, in which case a baculovirus expression system may be appropriate. Another alternative host is a mammalic cell line, for example Chinese Hamster ovary cells.

A DNA sequence encoding the PTPase of the present invention, or its functional derivatives, may be recombined with vector DNA in accordance with conventional techniques, including blunt-ended or staggered-ended termini for ligation, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and ligation with appropriate ligases. Techniques for such manipulations are disclosed by Maniatis et al.. supra. and are well known in the art.

As stated above, a suitable expression vector contains transcriptional and translational regulatory information able to direct and control gene expression in an appropriate host. These sequences are operably linked to the gene sought to be expressed and include a promoter (which directs the initiation of RNA transcription) , the Shine-Dalgarno sequence, capping sequence, CAAT sequence and the like (which are involved with initiation of transcription and translation) . The promoter sequences of the present invention may be either prokaryotic, eukaryotic or viral. Examples of suitable prokaryotic sequences include the P_R and P_L promoters of bacteriophage lambda (The Bacteriophage Lambda . Hershey, A.D., Ed., Cold Spring Harbor Press, Cold Spring Harbor, NY (1973); Lambda II. Hendrix, R.W. , Ed., Cold Spring Harbor Press, Cold Spring Harbor, NY (1980) ; the trp. recA. heat shock, and lacZ promoters of E. coli and the SV40 early promoter (Benoist, C. et al. Nature 290:304-310 (1981)).

As far as the Shine-Dalgarno sequence is concerned. preferred examples of suitable regulatory sequences are represented by the Shine-Dalgarno of the replicase gene of the phage MS-2 and of the gene ell of bacteriophage lambda. The Shine-Dalgarno sequence may be directly followed by the DNA encoding PTP35 and result in the expression of the mature PTP35 protein.

Alternatively, the DNA encoding PTP35 or genetic variants thereof may be preceded by a DNA sequence encoding a carrier peptide sequence. In this case, a fusion protein is produced in which the N-terminus of PTP35 is fused to a carrier peptide, which may help to increase the protein expression levels and intracellular stability, and provide simple means of purification. A preferred carrier peptide includes one or more of the IgG binding domains of Staphylococcus protein A. Fusion proteins comprising IgG binding domains of Protein A are easily purified to homogeneity by affinity chromatography e.g. on IgG- coupled Sepharose. A DNA sequence encoding a recognition site for a proteolytiϋ enzyme such as enterokinase, factor X or procollagenase may immediately precede the sequence for PTP35 or a variant thereof, to permit cleavage of the fusion protein to obtain the mature PTP35 protein.

Furthermore, a suitable expression vector includes an appropriate marker which allows the screening of the transformed host cells. Such marker- is typically an antibiotic resistance gene which confers to the transformed host the ability to grow on a selective medium containing the antibiotic to which the gene confers resistance.

The transformation of the selected host is carried out using any one of the various techniques well known to the expert in the art described in Maniatis et al, supra.

A further embodiment of the present invention is therefore a suitable expression vector containing the DNA sequence encoding the PTPase of the invention or a functional derivative thereof. The DNA sequence encoding PTP35 or variants thereof may be preceded by a sequence encoding a carrier peptide to obtain a fusion protein.

One further embodiment of the invention is a host cell, either prokaryotic or eukaryotic, transformed with the said expression vector and able to produce, under appropriate culture conditions, the PTPase of the invention or a functional derivative thereof.

The present invention is also directed to a process for preparing the PTPase protein of the invention or a functional derivative thereof, comprising:

(i) providing a sutiable host under such conditions as to obtain expression of the PTPase or functional derivative thereof, and (ii) purifying the thus obtained PTPase or functional derivative thereof.

The host is suitably prepared by:

(a) isolating a . DNA molecule consisting essentially of a nucleotide sequence encoding the PTPase or functional derivative thereof;

(b) inserting the said DNA molecule in a suitable expression vector; and (c) transforming an appropriate host with the said expression vector.

The PTPases and functional derivatives of the invention are useful in methods for testing of compounds capable of enhancing or inhibiting the phosphatase activity. The invention is also useful in diagnosing disorders involving the PTPase of the invention.

The ability of a compound under test to modify phosphatase activity can be tested in an in vitro system wherein the test compound is added to purified PTPase protein or enzymatically active derivatives thereof, and the effects on enzyme activity measured using standard enzymological procedures well known to those of skill in the art. Alternatively, the action of a compound on PTPase activity can be measured in a whole cell preparation using live or fixed cells, or a membrane fraction derived from live or fixed cells. This method is useful for screening compounds acting via the extracellular receptor portion of the protein, as well as compounds acting directly on the enzymatic portion of the protein. A test compound is incubated with cells, or with a membrane preparation derived therefrom, which express high amounts of the PTPase of this invention, such as NIH-3T3 cells.

The amount of cellular phosphotyrosine is then measured, using methods well-known in the art (Honegger, A.M. et al.. Cell 51:199-209 (1987); Margolis, B. et al.. Cell 57:1101- 1107 (1989)) . The results are compared to results obtained in the absence of the test compound, or in the absence or presence of a known activator of R- TPase. In such studies, the action of the test compound in the presence of an activator of tyrosine kinase can also be measured.

A compound which stimulates PTPase activity will result in a net decrease in the amount of phosphotyrosine, whereas a compound which inhibits PTPase activity will result in a net increase in the amount of phosphotyrosine.

In the case of growth factor receptors which are tyrosine kinases, such as the receptors for epidermal growth factor (EGF) and for platelet-derived growth factor (PDGF) , tyrosine phosphorylation is linked to cell growth and to oncogenic transformation. Activation of a PTPase, leading to dephosphorylation, would serve as a counterregulatory mechanism to prevent or inhibit growth, and might serve as an endogenous regulatory mechanism against cancer. Thus, mutation or disregulation of this receptor/enzyme system may promote susceptibility to cancer.

The insulin receptor is also a tyrosine kinase, and phosphorylation of tyrosine in cells bearing insulin receptors would be associated with normal physiological function. In contrast to the case of cell growth and cancer, activation of a PTPase would counteract insulin effects. Subnormal PTPase levels or enzymatic activity would act to remove a normal counterregulatory mechanisms.

Perhaps more "important, though, over-activity, or inappropriate activation, of a PTPase would be expected to inhibit or totally prevent the action of insulin on cells, leading to diabetes (of an insulin-resistant variety) .

Thus, susceptibility to diabetes may be associated with PTPase disregulation. In addition, recognition of tyrosine phosphatases expressed in pancreatic islets as autoantigens may lead to diabetes as a consequence of cells distruction by an autoimmune mechanism. In this case, the use of recombinant extracellular domain of such phosphatases may be used to block the binding of autoantibodies to phosphatases expressed on the surface of islet cells, thus providing a direct therapeutic effect.

Therefore, the methods of the present invention for measuring the amount or activity of PTPase associated with a cell or tissue, can serve as methods for identifying susceptibility to cancer, diabetes, or other diseases associated with alterations in cellular phosphotyrosine metabolism. In view of the specific expression of PTP35 in brain, particularly in differentiating central nervous system cells, regulation of PTP35 activity may be important in neuronal cells differentiation and survival. Since it has been reported that insulin receptors distribution in brain corresponds to location of proteins phosphorylated in tyrosine residues, the action of tyrosine phosphatases which are specifically expressed in brain, such as PTP35, may be important in the regulation of the insulin receptor tyrosine kinase in this tissue.

In view of the above considerations, an embodiment of the present invention is a method for identifying a compound capable of stimulating or inhibiting the enzymatic activity of a protein according to the invention, comprising:

(a) contacting the compound with the protein in pure form, in a membrane preparation, or in a whole live or fixed cell;

(b) incubating the mixture in step (a) for a sufficient interval;

(c) measuring the enzymatic activity of the protein;

(d) comparing the enzymatic activity to that of the protein incubated without the compound, thereby determining whether the compound stimulates or inhibits the activity.

The invention includes an antibody capable of binding to a PTPase or derivative thereof according to the invention. The antibody may be monoclonal or polyclonal. The antibody is useful for diagnosing disorders involving a PTPase of the invention.

Methods of producing monoclonal and polyclonal antibodies are well known. A method for producing a polyclonal antibody comprises immunising a suitable host animal, for example an experimental animal, with a PTPase or derivative of the invention and isolating immunoglobulins from the immune serum. The animal may therefore be inoculated with the PTPase or derivative, blood subsequently removed from the animal and the IgG fraction purified. A method for producing a monoclonal antibody comprises immortalising cells which produce the desried antibody. Hybridoma cells may be produced by fusing spleen cells from an inoculated experimental animal with tumour cells (Kohler and Milstein, Nature 256, 495-497, 1975).

An immortalized cell producing the desired antibody may be selected by a conventional procedure. The hybridomas may be grown in culture or injected intraperitoneally for formation of ascites fluid or into the blood stream of an allogenic host or immunocompromised host. Human antibody may be prepared by in vitro immunisation of human lymphocytes, followed by transformation of the lymphocytes with Epstein-Barr virus.

For the production of both monoclonal and polyclonal antibodies, the experimental animal is suitably a goat, rabbit, rat or mouse. If desired, the PTPase or derivative of the invention may be administered as a conjugate in which the PTPase or derivative is coupled, for example via a side chain of one of the amino acid residues, to a suitable carrier polypeptide. The carrier molecule is typically a physiologically acceptable carrier. The antibody obtained may be isolated and, if desired, purified.

The invention includes a method for the determination of a PTPase or derivative of the invention in a test sample, which method comprises contacting the test sample with an antibody of the invention, and determining the amount of antibody binding to the PTPase or derivative thereof. This method is useful in diagnosing disorders involving a PTPase of the invention. An ELISA (enzyme-linked immunoassay) method, such as a non-competitive ELISA, is suitably used for the determination. Typically, an ELISA method comprises the steps of (i) immobilising on a solid support an unlabelled antibody in accordance with the invention, (ii) adding a test sample containing the PTPase or derivative thereof to be determined such that the PTPase or derivative thereof is captured by the unlabelled antibody, (iii) adding an antibody in accordance with the invention which has been labelled, and (iv) determining the amount of bound labelled antibody.

Examples of suitable antibody labels include biotin (which may be detected by avidin conjugated to peroxidase) and alkaline phosphatase. A Western blotting method may be used for determining a PTPase or derivative of the invention. Such a method can comprise the steps of

(i) subjecting a test sample containing a PTPase or derivative thereof to gel electrophoresis,

(ii) transferring the separated proteins in the gel onto a solid support (e.g. a nitrocellulose support) by blotting, and (iii) allowing an antibody in accordance with the invention which has been labelled to bind to the PTPase or derivative thereof.

The invention includes a method of determination of an mRNA encoding a PTPase or derivative thereof according to the invention in a test sample, which method comprises hybridising mRNA present in the test sample with a DNA probe consisting essentially of sequence shown in Figure 1, and determining the amount of resulting DNA:mRNA hybrids.

Suitably, a Northern Blotting method is used for determination of mRNA, which method may comprise (i) subjecting a test sample containing an mRNA encoding a PTPase or derivative thereof according to the invention to gel electrophoresis,

(ii) transferring the separated RNA molecules in the gel onto a solid support (e.g. a nitrocellulse support) by blotting, (iii) allowing a DNA probe to hybridise to the mRNA, and (iv) measuring the amount of hybridised DNA probe.

Having now generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration and are not intended to be limiting of the present invention. In the accompanying drawings:

Fig.l Sequence of PTP35 The sequences of the cDNA clones PTP35,11 (Figure la) and PTP35, 12 (Figure lb) are shown together with translation of the major open reading frame. DNA sequence: the putative ATG (clone 11) and CTG (clone 12) start codons, and TGA termination codons are boxed. Protein sequence: the RGD feature, the putative transmembrane peptide and the tyrosine phosphatase consensus motif are boxed.

Fig.2 Panel A Northern blot analysis of PTP35 in different mouse tissues 20 μg of total RNA from mouse tissues was subjected to Northern blot analysis, using a Sall-EcoRI fragment from plasmid PTZ-PTP35 as a probe. Lane 1, liver; lane 2, spleen; lane 3, heart; lane 4, brain. The position of the 28S and 18S ribosomal RNA is indicated. A photography of the gel prior to RNA transfer blot is shown to demonstrate equal loading of the lanes. Panel B Expression of PTP35 in rat brain 10 μg of total RNA from rat cerebellum granules neurones at day 2 (lanel) and day 8 (lane 2) of culture were analyzed by Northern blot analysis, using a Sall- EcoRI fragment from plasmid PTZ-PTP35 as a probe. The position of the 28S and 18S ribosomal RNA is indicated. Equal loading of the lanes was verified by rehybridization of the same filter with a glyceraldeide-3-phosphate dehydrogenase (GAPDH) probe. Fig.3 Panel A Growth curve of Swiss NIH3T3 cells 3T3 cells were seeded at 10 3 cells/cm2ε.and cultured in DMEM medium supplemented with 10%FCS. Each dayiscells were trypsinized and counted. Panel

B Time-course of PT35 mRNA levels during cell growth 20 μg of total RNA from cells harvested at each day of culture were analyzed by Northern blot hybridization, using a Sall-EcoRI fragment from plasmid PTZ-PTP35 as a probe. The position of the

28S and 18S ribosomal RNA is indicated. Equal loading of all lanes was verified by rehybridization with a with an actin probe.

Fig. 4 Panel A Kinetics of induction of PTP35 mRNA in 3T3 cells by bFGF Northern blot hybridization of total RNA from cells untreated (-) or treated with 30 ng/ml of bFGF (+) at different times after treatment with bFGF. PTP35 mRNA is maximally induced 24 hours after bFGF addition. Equal loading of all lanes was verifyed by rehybridization with an actin probe. The position of 28S and 18S ribosomal RNA is indicated. Panel B Specificity of induction of PTP 35 mRNA Northern blot hybridization of total RNA 24 hours after treatment. . Lane 1, control; lane 2, addition of 30 ng/ml bFGF; lane 3, addition of 30 ng/ml bFGF + 10 μg/ml cycloheximide; lane 4, addition of 10 μg/ml cycloheximide; lane 5, addition of 10 ng/ml PDGF; lane 6, addition of 10 ng/ml PDGF + 10 μg/ml cycloheximide. Equal loading of all lanes was verified by rehybridization with an actin probe.

Fig. 5. Expression of PTP 35 extracellular domain

10% SDS-PAGE showing total cell extracts of HB101 cells transformed with pRIT33 (lane 1) , with pRIT33-PTP35XC (lane 2) or non-transformed (lane 3) . The position of bands corresponding to protein A (about 33 kDa) and protein A-PTP35XC (about 75 kDa) is indicated.

Fig. 6 Expression of PTP35 intracellular domain 10% SDS-PAGE showing expression and purification of

PTP35 intracellular domain. Soluble cell extract after sonication was incubated with gluthatione agarose resin, extensively washed and then incubated with thrombin. Essentially pure, soluble PTP35 intracellular domain was collected in the medium after release from the resin. Lane 1, molecular weight markers; lane 2, soluble cell extract; lane 3, washing medium; lane 4, aliquot of the resin after thrombin addition; lane 5, soluble pure PTP35 intracellular domain. The arrows indicate GST-PTP35 fusion (lane 2) or mature PTP35 intracellular domain (lane 5) .

EXAMPLE I

Isolation and Analysis of Murine PTP35 cDNA clones.

Polymerase Chain Reaction Amplification and Identification of PTP35 clone

Total RNA from actively growing Swiss 3T3 fibroblasts (ATCC CCL 92) was prepared according to standard procedures (Maniatis, T. , Frisch, E'.F. and Sambrook, J.: Molecular cloning. A labouratory manual. Cold Spring Harbour Labouratory, Cold Spring Harbour, NY 1989). First-strand cDNA was synthesized from 10 μg of total RNA using AMV reverse transcriptase (Boehringer Mannheim) and amplified by Polymerase Chain Reaction (PCR) using two degenerated primers encoding conserved phosphatase sequences. The sense primer 1 (5' AAG CTT CTG CAG GTC GAC TTT/C TGG GAA/G ATG GT/A/C/T/G TGG GA 3') harboured a Sail restriction site followed by a degenerated sequence encoding the conserved amino acids FWEMVWE. The complementary primer 4 (5' CGA ATT CGG TAC CGG ATC CTG CCC CGG CAC TGC AGT GCA C 3') harboured a sequence complementary to conserved amino acids VHCSAGA followed by an EcoRI restriction site. The two primers were used to amplify first strand cDNA from growing 3T3 cells using Taq Polymerase (Perkin-Elmer) . The annealing temperature was 55 C° and the polymerization reaction was done at 72 C°. 40 cycles of amplyfication were performed using a Perkin-Elmer thermal cycler.

The amplified fragment, with the expected size of about

360 bp, was cut with Sall/EcoRI and subcloned into plasmid pTZ19R

(commercially available from Pharmacia; see also Protein Engineering 1 , 67 (1986), and Proc. Natl. Acad. Sci 85_/ 4832 (1988)) cut with the same enzymes. Sequence of the different recombinant plasmids was performed directly on double strand DNA using Sequenase (United States Biochemicals) . The DNA of plasmid pTZ-PTP35 was prepared by standard procedures and cut with Sail and Eco RI enzymes. A 360 bp fragment, corresponding to the phosphatase DNA insert, was purified by gel electrophoresis followed by electroelution of the band as described (Maniatis, T. , Frisch, E.F. and Sambrook, J.: Molecular cloning. A labouratory manual. Cold Spring Harbour Labouratory, Cold Spring Harbour, NY 1989) .

Library screening

To obtain the complete DNA sequence of PTP35, an NIH Swiss 3T3 fibroblasts cDNA Lambda Zap II library (Stratagene, LaJolla, USA) was screened at high stringency by overnight hybridization at 65 *C in 5XSSC, 5X Denhardt's, 0.1%SDS, 250 mg/ml salmon sperm DNA. The 360 bp Sall/EcoRI fragment derived from plasmid pTZ-PTP35, which had been 32P-labeled using the random-priming method, was used as a probe at 2 x 10 cpm/ml.

5 Washing was performed at 65 ^βC in 1XSSC, 0.1%SDS. Out 2 X 10 clones, 7 positives were selected and furtherly characterized.

DNA of the recombinant pBluescript SK~ plasmids was excided from the selected clones as described by the manufacturer (Stratagene, La Jolla, USA) and subjected to EcoRI digestion, to verify the presence and size of the inserts. Double stranded DNA of the different clones was directely sequenced using the dideoxynucleotide chain termination method (Sequenase, United States Biochemical; Proc. Natl. Acad. Sci 24. 5463-5476 (1977)) and the universal forward and reverse primers. The relative order and orientation of the EcoRI fragments in the recombinant plasmids was obtained by restriction mapping and sequence overlapping. All regions were sequenced in both orientations using internal sequencing primers. All the sequenced clones were found to contain portions of the same sequence. The sequence of the fragment obtained by PCR was also confirmed. The sequences of the two longest clones, PTP35,11, and PTP35, 12 are shown in Figure la and lb.

Analysis of the sequence of clone PTP35, 11

Nucleotide sequence analysis was performed using the Geneworks program (Intelligenetics, CA) . Translation of the sequence contained in the cDNA clone PTP35,11 revealed the existance of a major open reading frame encoding 790 amino acids, assuming that translation initiates at nucleotide 505 (an in- frame stop codon is present at nucleotide 124, 381 nucleotides upstream) . On the basis of the presence of a hydrophobic stretch between amino acids 388 and 408, the protein is classified as transmembrane. The predicted intracellular portion of the protein consists of a unique domain displaying significant homology to the intracellular catalytic regions of the previously described transmembrane phosphatases. The conserved sequence VHCSDG, which has been proposed to be part of the phosphatase catalytic site is present in PTP35. Since there is no duplication of the catalytic domain, protein PTP35 belongs to tyrosine phosphatases Class II.

Analysis of the sequence of clone PTP 35.12

Nucleotide sequence analysis of clone PTP35,12 was performed using the Geneworks program (Intelligenetics, CA) . The sequence was found to correspond to that of clone 11 of PTP35 from nucleotide 279 to the end, while the two sequences diverge at their 5' ends, possibly due to alternative splicing.

Translation of the sequence contained in the cDNA clone PTP35,12 revealed the existance of a major open reading frame encodoing 961 amino acids, assuming that translation initiates with the CTG codon at nucleotide 157 (an in frame stop codon is present at nucleotide 99, 58 nucleotides upstream) . On the basis of the presence of a hydrophobic stretch between amino acids 559 and 579, the protein is classified as transmembrane. The putative extracellular domain encodes 558 amino acids and includes a short hydrophobic sequence of 17 amino acids which is recognized by the computer as an eukaryotic secretory signal sequence. The mature PTP35 molecule encoded in clone PTP35,12 would then start at amino acid 18. The predicted intracellular portion, which corresponds to that encoded in clone PTP35,11, consists of a unique domain displaying significant homology to the intracellular catalytic regions of the previously described transmembrane phosphatases. The sequence IIVHCSDGAGRTG at amino acids 886 to 899 matches the consensus sequence for PTPase activity.

EXAMPLE II Analysis of PTP35 mRNA expression PTP35 mRNA distribution in tissues

Northern blot analysis on total RNA from mouse spleen, heart, brain and liver (purchased from Clonetech, CA) was performed according to standard procedures (Maniatis, T. , Frisch, E.F. and Sambrook, J. : Molecular cloning. A labouratory manual. Cold Spring Harbour Labouratory, Cold Spring Harbour, NY 1989) , using 20 μg of RNA per lane. Hybridization was performed overnight at 42^βC in 50% formamide, 5 X SSC, 1 X Denhardt's, 0.1%SDS and 250 mg/ml of salmon sperm DNA, using the Sall/EcoRI fragment used for the library screening as a probe at 2 x 10 cpm/ml. Washing was done at 42°C in 1 X SSC, 0.1% SDS. Higher stringency washing did not noticeably affect the hybridization pattern.

Northern analysis (Fig. 2, panel A) revealed a highly specific pattern of expression for PTP35 in mouse, essentially restricted to the brain. In this tissue, an mRNA of about 3200 bp was expressed at very high level, in addition to a second mRNA of smaller size, about 1400 bp, expressed in lower amounts. Analysis of PTP35 mRNA regulation

On the basis of the specific expression of PTP35 in mouse brain, the expression of PTP35 mRNA in rat cerebellum granules neurons was investigated. Primary cells were isolated from rat brain tissue as described (A. Frandsen and A. Schousboe: Int. J. of Dev. Neurosci, 8. (2): 209-216, 1990). Cells were seeded at 3 X 10 cells per dish in 35 mm dishes and cultured for 8 days. Cells at day 2 (poorly differentiated) and day 8 (advanced differentiation state) were harvested and total RNA was prepared and subjected to Northern blot hybridization as described (Maniatis, T., Frisch, E.F. and Sambrook, J.: Molecular cloning. A labouratory manual. Cold Spring Harbour Labouratory, Cold Spring Harbour, NY 1989) . Hybridization and washing conditions were as usual (see the above paragraph) . The Sall/EcoRI PTP35 fragment was used as a probe at 2 x 10 cpm/ml. As shown in fig. 2 B, rat cerebellum granules neurons express high levels of PTP35 mRNA of the same size, about 3200 bp, of that observed in mouse brain. Expression is higher in cells in advanced differentation state, with extended neurite outgrowth and cell to cell connnections, suggesting that PTP35 is involved in rat cerebellum granules neurons differentiation and survival.

The regulation of PTP35 mRNA expression in growing NIH3T3 cells

3 was analyzed. NIH3T3 cells were seeded at low density, 10 cells per cm , and cultivated for 7 days in DMEM medium (Gibco) supplemented with 10% Fetal Calf Serum (FCS) (Gibco) . Medium was changed every other day. Each day, cells from two dishes were trypsinized and counted. The growth curve obtained for 3T3 cells is shown in figure 3, panel A. In parallel, each day cells were harvested and total RNA was prepared and subjected to Northern blot analysis as described (Maniatis, T. , Frisch, E.F. and Sambrook, J.: Molecular cloning. A labouratory manual. Cold Spring Harbour Labouratory, Cold Spring Harbour, NY 1989) , using 20 μg of RNA per lane. Hybridization was performed under standard conditions (see the above paragraph) using the Sall/EcoRI PTP35 fragment as a probe at 2 x 10 cpm/ml. The results of the Northern blot hybridization are shown in fig. 3, panel B. A mRNA of the same size, about 3200 bp, of the predominant signal observed in mouse and rat brain was seen. Analysis of mRNA steady-state levels obtained from cells at different days showed that PTP35 mRNA expression was directly correlated with cell growth. mRNA maximum levels were observed in actively growing cells and decreased until little or no expression was observed in confluent, quiescent 3T3 cells, suggesting that PTP35 is involved in the regulation of cell growth.

The effect of basic Fibroblast Growth Factor (bFGF) stimulation on PTP35 mRNA levels in NIH3T3 cells was examined. Cells were seeded at semiconfluence (3 X 10⁴ cells/cm ) in DMEM+10% FCS and then incubated in DMEM medium + 0.4 % FCS for 24 hours. bFGF at 30 ng/ml was then added and, at regular intervals, cells were harvested in parallel with untreated control cells. Total RNA was prepared and subjected to Northern blot analysis as described (Maniatis, T. , Frisch, E.F. and Sambrook, J.: Molecular cloning. A labouratory manual. Cold Spring Harbour Labouratory, Cold Spring Harbour, NY 1989) , using PTP35 Sall/EcoRI fragment as a probe. Hybridization conditions were the same as usual. As shown in fig. 4, PTP35 mRNA steady-state levels were increased after bFGF stimulation and reached a peak after 24 hours. This effect was not inhibited when bFGF stimulation was performed in the presence of 10 μg/ml of cycloheximide. The same effect was observed after Platelet Derived Growth Factor (PDGF) and serum stimulation.

EXAMPLE III

Expression of PTP35 in prokaryotic cells

The vector pRIT33 (commercially available from Pharmacia; see also Methods in Enzymology 185. 144-161 (1990)) is a derivative of plasmid pRIT2 and is designed for temperature- inducible expression of intracellular hybrid proteins in E. coli after induction of lambda P_R promoter. Alternatively, expression can be obtained by induction of the lacUV5 promoter, which is inserted up-stream of the lambda P_R promoter, with 1 mM IPTG. The construct contains the IgG-binding domains of Staphylococcal protein A which are under the control of the lambda P_ promoter. A multiple cloning site facilitates the insertion of foreign genes at the 3' side of protein A. The sequence encoding the recognition site for Factor X is present at the end of the Protein A coding sequence.The protein A transcription termination sequence is inserted immediately donwstream from the multiple cloning site. To obtain a Protein A-PTP35 fusion protein, the sequence encoding PTP35 protein was inserted into plasmid pRIT33. The PTP35 sequence was retrieved from plasmid PTP35,4 which is one of the clones isolated after the screening of the library described in Example 1. PTP35,4 corresponds to plasmid pBluescript SK^" containing the PTP35 coding sequence oriented in such way that its 5' end is preceded by the BamHI site of the polylinker and its 3' end is followed by the Sail site of the polylinker. A Stul-Sall fragment of about 3000 bp encompassing PTP35 coding sequence (nucleotides 590 to 3561 of clone 12, Figure lb) was isolated from plasmid PTP35, 4 and subcloned into plasmid pRIT33 cut with Smal and Sail. Smal and StuI blunt ends ligated together resulted in appropriate in-frame positioning of the PTP35 sequence immediately after the Factor X recognition sequence in pRIT33. The resulting plasmid pRIT33-PT35 carries an open reading frame, coding for a fusion protein of 120 kD, composed of protein A at the N-terminus followed by the PTP35 protein, separated be a specific sequence for proteolytic cleavage.

Alternatively, a portion of the extracellular domain of PTP35, encompassing 397 amino acids, was separately expressed in E. coli as a Protein A fusion protein. To isolate the DNA sequence encoding this domain, plasmid PTP35,4 was cut with Ncol and the protruding ends were filled in by Klenow DNA polymerase to leave blunt ends. The plasmid was then cut with StuI and a 1200 bp fragment with blunt extremities (corresponding to nucleotides 590 to 1804 of clone PTP35,12) was subcloned into pRIT33 cut with Sma I and dephosphorylated. The resulting plasmid pRIT33-PTP35XC carries an open reading frame encoding a protein of about 75 kD composed of the protein A followed by the PTP35 extracellular domain amino acids, separated by a specific sequence for proteolytic cleavage. Expression of the Protein A-PTP35XC fusion protein was analyzed in HB101 E.Coli cells (J. Mol. Biol. 41, 454, 1969) cotransformed with a compatible plasmid, named pRITcl857, harbouring a termosensitive repressor of lambda P_R promoter (Nilsson, B. and Abrah sen, L. Methods in Enzymology 185. 144- 161 , 1990 ) .

Induction was obtained by a temperature shift (heat shock) from 30^βC to 42^βC, whereby the repressor is inactivated and transcription of the promoter is induced. Alternatively, expression was obtained after induction of the lacUV5 promoter with 1 mM IPTG. Since this promoter is never completely silent, high levels of protein are also expressed by extended growth of the transformed clones at 30". An example of the fusion protein levels after a 3 days growth at 30^β is shown in Figure 5. When grown in these conditions the amount of the Protein A-PTP35XC fusion protein represented around 20% of the total cellular protein. The apparent M_r of the hybrid protein is, as expected, 75 Kd on SDS-PAGE.

EXAMPLE IV

Expression of PTP35 phosphatase domain in prokaryotic cells

Plasmids of the pGEX series (commercially available from Pharmacia; see also Smith, D. and Kevin, J. (1988) Gene 67, 31-40) have been designed for the expression of recombinant proteins in E. coli as fusions with the C-terminus of Sj26, a 26- kD glutathione-S-transferase (GST) encoded by the parasitic helminth Schistosoma iaponicum. These fusion proteins can be purified from crude bacterial lysates under non-denaturing conditions by affinity chromatography on immobilized glutathione. Moreover, the vectors have been engineered so that the GST carrier can be cleaved from fusion proteins by digestion with site-specific proteases such as thrombin, yielding the mature recombinant polypeptide of interest in a practically pure form.

To obtain a GST-PTP35 PTPase domain, the sequence encoding the putative intracellular domain (amino acids 583-961 of the protein encoded in clone PTP35,12) was amplified by PCR with 5' and 3' primers containing BamHI and EcoRI sites, respectively. The sequence was then inserted into plasmid pGEX- 2T cut with the same enzymes. The resulting plasmid, named pFC221, encodes a fusion protein of about 70 kD, composed of GST at the amino terminus followed by the putative PTPase domain of PTP35. High level expression of the fusion protein in the DH5α strain after IPTG induction was obtained as described (Smith, D. and Kevin, J. (1988) Gene 67, 31-40). For PTP35 PTPase domain purification, the soluble lysate after cell sonication was incubated with agarose-glutathione resin (Pharmacia, Sweden) . After incubation, the resin was extensively washed to eliminate non-specific binding and incubated with a catalytic quantity of thrombin to release the PTPase domain from the GST moiety bound to the resin. The incubation medium containing the soluble PTPase domain was then collected, 40% glycerol was added and the enzyme was kept frozen in aliquots at -80°C. An example of GST- PTP35 PTPase domain expression and purification on agarose- glutathione is shown in Fig.6.

Claims

1. A mammalian receptor-type protein tyrosine phosphatase (PTPase) whose, extracellular domain includes an Arg- Gly-Asp sequence, or a functional derivative thereof.

2. A PTPase according to claim 1 of natural origin and substantially free of other proteins with which the PTPase is associated in nature.

3. A PTPase according to claim 1 or 2 having substantially the amino acid sequence shown in Figure la or lb, or a functional derivative thereof.

4. A fusion protein comprising a PTPase or functional derivative thereof according to claim 1, 2 or 3, fused to a carrier polypeptide.

5. A DNA molecule consisting essentially of a nucleotide sequence encoding a PTPase or functional derivative thereof according to claim 1.

6. A DNA molecule according to claim 5 which is a cDNA molecule.

7. A DNA molecule according to claim 5 which is a genomic DNA molecule.

8. A DNA molecule according to any one of claims 5 to 7 wherein a nucleotide sequence encoding a carrier polypeptide is linked to the said sequence encoding a PTPase or functional derivative thereof.

9. A fusion protein according to claim 4 or a DNA molecule according to claim 8 wherein the carrier polypeptide includes one or more of the IgG binding domains of Staphylococcus protein A.

10. An expression vector containing a DNA molecule according to any one of claims 5 to 9.

11. A host transformed with an expression vector according to claim 10.

12. A host according to claim 11 which is a eukaryotic host.

13. A host according to claim 11 which is a prokaryotic host.

14. A host according to claim 13 which is E. coli.

15. A process for preparing a PTPase or functional derivative thereof according to claim 1, which process comprises:

(i) providing a host according to any one of claims 11 to

14 under such conditions as to obtain expression of the

PTPase or functional derivative thereof; and (ii) purifying the thus obtained PTPase or functional derivative thereof.

16. A process according to claim 15, wherein the said host is prepared by:

(a) isolating a DNA molecule consisting essentially of a nucleotide sequence encoding the said PTPase or functional derivative thereof;

(b) inserting the said DNA molecule into a suitable expression vector; and

(c) transforming an appropriate host with the said expression vector.

17. A method for identifying a compound capable of stimulating or inhibiting the enzymatic activity of a PTPase or functional derivative thereof according to claim 1, 2 or 3, which method comprises: (a) contacting the compound with the PTPase or functional derivative thereof in pure form, in a membrane preparation or in a whole live or fixed cell;

(b) incubating the mixture of step (a) for a sufficient period of time; and

(c) comparing the enzymatic activity of the mixture of step (a) with that of the PTPase or functional derivative thereof incubated without the compound, thereby determining whether the compound stimulates or inhibits the activity.

18. A polyclonal or monoclonal antibody capable of binding to a PTPase or derivative thereof according to claim 1, 2 or 3.

19. A method of determination of a PTPase or derivative thereof according to claim 1 in a test sample, which method comprises contacting the test sample with an antibody according to claim 18, and determining the amount of antibody binding to the PTPase or derivative thereof.

20. A method of determination of an mRNA encoding a PTPase or derivative thereof according to claim 1 in a test sample, which method comprises hybridising mRNA present in the test sample with a DNA probe consisting essentailly of sequence shown in Figure 1, and determining the amount of resulting DNA:mRNA hybrids.