WO1996012018A2 - Novel immunophilins and corresponding nucleic acids - Google Patents

Abstract

The present invention relates to novel immunophilin proteins predominently expressed by T-cells, belonging to the FKBP class, capable of binding to FK-506 (or its analogs) and to rapamycin, inhibiting calcineurin phosphatase activity when bound to ligand, and whose mRNA is induced by glucocorticoids (e.g. dexamethazone). These proteins have a peptidyl-prolyl cis/trans isomerase activity. Also included in the invention are the nucleic acids coding for said immunophilins, antibodies against these immunophilins and uses therefore, for instance in the identification/screening of compounds useful as immunosuppressive agents.

Description

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NOVEL IMMUNOPHILINS AND CORRESPONDING NUCLEIC ACIDS

FIELD OF THE INVENTION

The present invention relates to novel immunophilin proteins and uses therefor. For example, such proteins can be employed for identifying compounds useful as i munosuppressive agents. The present invention also relates to nucleic acids encoding the invention immunophilin proteins, antibodies specific for such proteins, and uses therefor.

BACKGROUND OF THE INVENTION

FK-506 and cyclosporin A (CsA) are potent immunosuppressive drugs that inhibit T-lymphocyte activation by selectively blocking transcription of early lymphokine genes. Drug action is mediated by binding to intracellular binding proteins. Although their effects upon T-lymphocyte activation are similar, FK-506 and CsA bind to distinct members of the immunophilin protein family. FK-506 binding proteins (FKBPs) are members of a class of immunophilins that form complexes with FK-506, as well as with the structurally related immunosuppressive drug, rapamycin. Cyclophilins are members of a class of immunophilins that form complexes with CsA.

The first FKBP to be described at the protein level (Harding et al., Nature 341:758-760 (1989); Siekerka et al., Nature 341:755-757 (1989)) and cDNA (Maki et al., Proc . Natl . Acad . Sci . USA 87:5440-5443 (1990); Standaert et al., Nature 346:671-674 (1990)) was FKBP12, an abundant cytosolic immunophilin of M_r 11,800. More recently, FKBP13 (Jin et al., Proc . Natl . Acad . Sci . USA 88:6677-6681 (1991)) and FKBP25 (Galat et al. , Biochemistry 31:2427-2434 (1991)) have been characterized as immunophilins that bind both FK-506 and rapamycin. Recently, FKBP59 has been characterized as an immunophilin that binds to a 90 kDa heat shock protein (hsp90) and associates with steroid receptor complexes (Ruff et al., J . of Biol . Chem . 267:21285-21288 (1992)). Even more recently, FKBP12.6 has been purified and characterized as the immunophilin most closely related to FKBP12 (Sewell et al., J . of Biol . Chem . 269:21094-21102 (1994)).

Of five FKBPs isolated and characterized, only two bind to FK-506 to form complexes which inhibit calcineurin. These are FKBP12 and FKBP12.6. The phosphatase activity of calcineurin is a critical factor in the signal transduction pathway that leads to the expression of early lympokine genes, and activation of T-lymphocytes. Accordingly, the identification of FKBPs which bind FK-506 and (when complexed with ligand) inhibit calcineurin, will enable development of compounds which inhibit T-lymphocyte activation by selectively blocking transcription of early lymphokine genes.

Thus FKBP12 and FKBP12.6 represent the only immunophilins to date that operate on a well defined pathway leading to clearly identified events that result in inhibition of T-cell activation. The identification of additional immunophilins which are involved in a well defined pathway leading to clearly identified events that result in inhibition of T-cell activation would be of great value in the development of compounds which inhibit transcription of early lymphokine genes.

BRIEF DESCRIPTION OF THE INVENTION

In accordance with the present invention there are provided novel immunophilin proteins. Invention proteins allow the identification of compounds useful as immunosuppressive agents that prevent T-cell activation.

Invention immunophilin proteins are characterized by being predominantly expressed in T-cells and capable of inhibiting calcineurin phosphatase activity when bound to ligand. The invention proteins are further characterized as corresponding to mRNAs induced by glucocorticoids.

In accordance with other aspects of the invention, there are provided nucleic acids encoding invention proteins and fragments thereof, antibodies specific for invention proteins and uses of invention nucleic acids and specific antibodies.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, there are provided isolated mammalian immunophilin proteins characterized as being expressed predominantly in T-lymphocytes and capable of inhibiting the phosphatase activity of calcineurin when complexed with ligands. Invention proteins are further characterized as corresponding to mRNAs induced by glucocorticoids. The proteins of the invention belong to a class of immunophilins designated FKBPs, i.e. they bind to the structurally related ligands FK-506 and rapamycin, as well as analogs thereof. Additionally, the present invention provides isolated glucocorticoid inducible nucleic acids encoding the mammalian immunophilin proteins of the invention.

Glucocorticoids and cyclic AMP induce dramatic biochemical and morphological changes in T-lymphocytes through unknown mechanisms. In order to identify genes involved in the glucocorticoid response, a cDNA library was prepared from poly (A) RNA isolated from a glucocorticoid-sensitive murine thymoma cell line treated with glucocorticoids and forskolin, an agent which increases intracellular levels of cAMP. The cell line used to construct the library is a thioguanine resistant derivative of a glucocorticoid-sensitive BALB/c murine thymoma cell line. The thioguanine resistant cell line, designated WEHI-7TG, and the glucocorticoid sensitive cell line, designated EHI-7, are described by Bourgeois and Newby, (Cell 11:423-430 (1977)) and Harris et al. , (J . Immunol . 110:431-438 (1973)).

The cDNA library, constructed in lambda vector, comprised 5x10 lambda recombinants. The library was screened with a subtracted cDNA probe enriched for sequences induced by glucocorticoid/forskolin treatment of WEHI-7TG cells. Positive cDNA clones corresponding to mRNAs induced by this treatment were subjected to Northern analysis to verify that the positive cDNA clones represented genes induced by glucocorticoid and/or forskolin.

Total cellular RNA for Northern analysis was obtained from untreated CXG56D3 cells, untreated WEHI-7TG cells, and WEHI-7TG cells treated with either dexamethasone, forskolin or both drugs in combination. CXG56D3 cells are a variant of EHI-7TG cells selected for resistance to cAMP and dexamethasone by a two-step procedure described by Gruol et al., (J . Biol . Chem . 261:4904-4914 (1986)).

DNA probes for Northern analysis were prepared from cDNA inserts obtained from the previously described lambda cDNA library. Radioactive probes representing each lambda cDNA insert were hybridized to Northern blots of total cellular RNA. Thirteen positive clones were identified. These cDNA clones correspond to genes regulated by glucocorticoid and cAMP in the EHI-7TG cell line.

Additional characterization of the positive clones was carried out by examining the tissue expression pattern of the corresponding mRNAs. Northern blots prepared with total RNA isolated from BALB/c mouse tissue were hybridized to probes prepared from cDNA inserts obtained from lambda recombinants previously described. Expression of genes corresponding to several of the clones showed specificity for lymphoid tissue. One clone, clone 213, hybridized strongly to mRNA in the thymus but showed no appreciable hybridization to mRNA in other tissues tested, namely brain, heart, kidney, liver, lung and spleen.

Clone 213 contained a cDNA insert of approximately 2.2kb. A radiolabeled probe, prepared from a fragment from the 5' end of the 2.2kb insert, was used to screen a murine thymus cDNA library for full length cDNAs. A clone of approximately 3.5kb was isolated. A second radiolabeled probe, prepared from a fragment from the 5' end of the 3.5kb clone, was used to rescreen the murine thymus cDNA library. A cDNA clone, designated 213-12A, was obtained upon rescreening the murine thymus cDNA library. Clone 213-12A represents a glucocorticoid inducible nucleic acid of the present invention. The open reading frame of clone 213-12A was sequenced in its entirety. The sequence of the coding strand, which is 2226 nucleotides, is set forth in SEQ ID NO:l. The amino acid sequence, from amino terminus to carboxyl terminus, is set forth in SEQ ID NO:2. The protein has 456 amino acid residues and an M_r of about 50,961.

In accordance with a further embodiment of the present invention, the receptor-encoding cDNAs described herein can be employed to probe library(ies) (e.g., cDNA, genomic, and the like) for additional sequences encoding novel immunophilins. Such screening is initially carried out under low-stringency conditions, e.g., a temperature of less than about 42°C, a formamide concentration of less than about 50%, and a moderate to low salt concentration. One such set of screening conditions comprise a temperature of about 37°C, a formamide concentration of about 20%, and a salt concentration of about 5X standard saline citrate (SSC; 2OX SSC contains 3M sodium chloride, 0.3M sodium citrate, pH 7.0). Such conditions will allow the identification of sequences which have a substantial degree of similarity with the probe sequence, without requiring perfect homology for the identification of a stable hybrid. The phrase "substantial similarity" refers to sequences which share at least 50% homology. Preferably, hybridization conditions will be selected which allow the identification of sequences having at least 70% homology with the probe, while discriminating against sequences which have a lower degree of homology with the probe.

Thus, for example, screening of a human thymus library, first with an 870 base pair fragment obtained from clone 213-12A (corresponding to nucleotides 463-1333 as shown in SEQ ID N0:1), then with a 561 base pair fragment derived from a different portion of clone 213-12A (corresponding to nucleotides 1-561 as shown in SEQ ID NO:l), led to the identification of a human clone corresponding to clone 213-12A. The nucleotide sequence of a full length human clone corresponding to clone 213-12A, and the deduced amino acid sequence thereof is set forth in SEQ ID NO:3. See also Examples 9-11 herein.

Isolated glucocorticoid inducible nucleic acids of the present invention comprise (a) nucleic acids encoding the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:4; (b) nucleic acids that hybridize to the nucleic acids of (a) wherein said nucleic acids encode biologically active immunophilin proteins; or (c) nucleic acids which are degenerate with respect to either (a) or (b) wherein said nucleic acids encode biologically active protein.

As employed herein, the phrase "nucleic acid" refers to ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) . DNA can be either complementary DNA (cDNA) or genomic DNA, e.g. a gene encoding an immunophilin protein of the invention.

The invention nucleic acids can be produced by a variety of methods well known in the art, e.g. the methods described in Examples 1, 2, 3 and 9, and by PCR amplification using oligonucleotide primers from various regions of SEQ ID NO:l or SEQ ID NO:3.

Nucleic acids described herein are particularly useful for producing invention immunophilin proteins when such nucleic acids are incorporated into protein expression systems such as are known to those of skill in the art. In addition, such nucleic acids or fragments thereof can be labeled with a readily detectable substituent and used as hybridization probes for assaying for the presence and/or amount of related genes or mRNA transcripts. The nucleic acids described herein, and fragments thereof, are also useful as primers and/or templates in PCR reactions for amplifying genes encoding the immunophilin proteins described herein.

Proteins contemplated for use in the practice of the present invention can be produced recombinantly in suitable hosts, e.g. E. coli and the like. Thus, for example, a cDNA encoding the open reading frame of an invention immunophilin protein, obtained by PCR amplification of plasmid DNA, can be subcloned into a suitable vector such as pGEM-3X (Pharmacia) . Following transformation into E . coli , expression of recombinant protein can be induced using isopropyl β-D-thiogalactopyranoside. The immunophilin protein product is isolated and purified utilizing affinity chromatography.

Alternatively, native immunophilin protein, corresponding to the recombinately produced invention protein, can be isolated from thymus cell extracts and purified using a drug affinity column. For example, rapamycin, a potent immunosuppressant known to bind invention proteins, can be affixed to an inert solid support according to the method of Fretz et al. (J. Am. Chem . Soc . 113:1409-1411 (1991)). The rapamycin affinity matrix can then be contacted with thymus cell extracts to obtain isolated native immunophilin protein, corresponding to invention protein.

Isolated immunophilin proteins of the present invention comprise protein having the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:4, as well as homologues and equivalents thereof (e.g. proteins which have substantially the same amino acid sequence and retain comparable functional and biological activity of the protein defined by the reference amino acid sequence) . The present invention further includes peptide fragments of invention proteins which fragments retain the functional and/or biological activity of invention proteins.

In accordance with another embodiment of the present invention, there are provided antibodies raised against invention immunophilin proteins. Such antibodies can be prepared employing standard techniques, as are well known to those of skill in the art, using invention proteins, or fragments thereof, as antigens for antibody production as described in Example 8. Antibodies of the present invention are typically produced by immunizing a mammal with an inoculum containing an invention immunophilin protein, or fragment thereof, thereby inducing the production of antibody molecules having immunospecificity for the immunizing agent. Invention antibodies can be used to block binding of ligands to invention immunophilin proteins and thereby reverse the inhibitory effect of such proteins on calcineurin phosphatase activity.

Antibodies to invention proteins can be obtained, for example, by immunizing New Zealand white rabbits with a suitable synthetic peptide fragment to which Tyr has been added at the C-terminus in order to couple it to KLH

(keyhole limpet he ocyanin) by a bisdiazotized benzidine

(BDB) linkage. Animals are immunized with the peptide coupled antigen and then bled two weeks post injection.

Antiserum is examined for its capacity to bind recombinant invention immunophilin protein in E. coli cells and endogenous immunophilin protein produced in EHI-7TG cells and thymus tissue. Antisera are then collected from the mammal and purified to the extent desired by well known techniques.

The enzymatic properties of the immunophilin proteins of the invention can be assessed using known methods. As described in Example 12, the assay of Fischer et al. (Nature 337:476-478 (1989)) was used to measure peptidyl prolyl cis-trans isomerization (PPIase) activity of recombinant immunophilin protein of the invention. Immunophilin proteins of the invention are active catalysts of the PPIase reaction which employs N-succinyl-Ala-X-Pro-Phe-p-nitroanilide (SEQ ID NO:5) as a substrate, where X is Leu, Phe, Val or Ala.

The PPIase activity of invention protein is inhibited by rapamycin and FK-520. The affinity of invention protein for these drugs indicates that the immunophilin proteins of the invention could bind to the drugs at systemic concentrations (blood levels) achieved during clinical use of these drugs.

In accordance with yet another aspect of the present invention, there is provided a competitive binding assay for identifying compounds which bind to immunophilin proteins of the invention. The assay is conducted by contacting invention immunophilin protein with labeled immunosuppressive compound such as, for example, labeled FK-506, which is known to bind invention immunophilin protein, in the presence or absence of test compound. Thereafter, protein bound compound and unbound compound are separated and the amount of label associated with protein in the presence of test compound relative to the amount associated with protein in the absence of test compound is quantified. Prescreening of potential candidate "ligands" for invention immunophilin proteins is preferably done with this binding assay prior to evaluation of such ligands in the calcineurin phosphatase inhibition assay.

The proteins of the invention are characterized by their ability, when complexed with immunosuppressive ligands, to inhibit calcineurin phosphatase activity. This inhibition is related to blockage of calcium associated events necessary for transcription of early lymphokine genes such as interleukin-2 (IL-2) . At any given time, most T-lymphocytes of the immune system are in a nonproliferating state. Following activation of a T-lymphocyte through interaction of a T-cell receptor and an antigen presenting cell, a Ca ^* dependent signal is generated. This Ca 2+ signal acti.vates calci.neuri.n, a protein phosphatase. Activated calcineurin dephosphorylates the T-cell transcription factor, NF-AT, thereby activating transcription of IL-2.

When proteins of the invention are complexed with suitable immunosuppressive ligands, such complexes have the ability to bind to calcineurin and inhibit its phosphatase activity. This results in the inhibition of lymphokine production which produces a compromised immune system.

Because invention immunophilin proteins are predominantly expressed in T-lymphocytes, such proteins can be utilized in screening for agents which are selective for T-cell mediated events. Agents identified by such methods should be less likely to exhibit adverse reactions than agents identified employing previously described immunophilin proteins which are ubiquitous in mammalian species.

In accordance with yet a further aspect of the present invention, there are provided methods for identifying compounds capable of binding to invention proteins and inhibiting the phosphatase activity of calcineurin. One such method comprises independently contacting labeled phosphorylated substrate with test compound, increasing amounts of invention protein and sufficient amounts of calcineurin and calmodulin to produce reaction mixtures containing free labeled phosphate. Free labeled phosphate is then determined in each reaction mixture. Test compounds capable of inhibiting calcineurin phosphatase activity will be those compounds which cause decreased production of free labeled phosphate in the presence of increasing amounts of invention protein.

Another such method comprises independently contacting labeled phosphorylated substrate with invention protein and with sufficient amounts of calcineurin and calmodulin to produce free labeled phosphate. Thereafter, the foregoing step is repeated in the further presence of test compound. The amount of free labeled phosphate generated in the presence of invention protein alone, relative to the amount generated in the further presence of test compound is determined. Test compounds capable of inhibiting calcineurin phosphatase activity will be those compounds which cause decreased production of free labeled phosphate.

Suitable labeled phosphorylated substrate which may be employed in the above-described methods includes

[ P]RII peptide which is a phosphorylated peptide fragment

(RII) from the regulatory subunit of cAMP-dependent kinase.

Other appropriate substrates containing other labeling moieties are well known to those skilled in the art to which this invention applies.

As used herein, the term FK-506 refers to 17-allyl-l, 14-dihydroxy-12-[2-(4-hydroxy-3-methoxycyclo- hexyl)-l-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetra- methyl-11, 28-dioxa-4-azatricyclo[22.3.1.0*'⁹]octacos- 18-ene-2,3,10,16-tetraone. The term FK-520 refers to 17-ethyl-l,14-dihydroxy-12-[2-(4-hydroxy-3-methoxycyclo- hexyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetra- methyl-11,28-dioxa-4-azazatricyclo[22.3.1.0⁴'⁹]octacos- 18-ene-2,3,10,16-tetraone.

Moderately stringent hybridization employed in the present invention refers to conditions that permit target-DNA to bind a complementary nucleic acid that has about 60%, preferably about 75%, more preferably about 85%, homology to the target DNA; with greater than about 90% homology to target-DNA being especially preferred. Preferably, moderately stringent conditions are conditions equivalent to hybridization in 50% formamide, 5X Denhart's solution, 5X SSPE, 0.2% SDS at 42°C, followed by washing in 0.2X SSPE, 0.2% SDS, at 65°C.

High stringency hybridization employed in the present invention refers to conditions that permit hybridization of only those nucleic acid sequences that form stable hybrids in 0.018M NaCl at 65°C (i.e., if a hybrid is not stable in 0.018M NaCl at 65°C, it will not be stable under high stringency conditions, as contemplated herein) . Preferable, high stringency conditions are conditions equivalent to hybridization in 50% formamide, 5X Denhart's solution, 5X SSPE, 0.2% SDS at 42°C, followed by washing in 0.1X SSPE, and 0.1% SDS at 65°C.

Low stringency hybridization employed in the present invention refers to conditions equivalent to hybridization in 10% formamide, 5X Denhart's solution, 6X SSPE, 0.2% SDS at 42°C, followed by washing in IX SSPE, 0.2% SDS, at 50°C. Denhart's solution and SSPE (see, e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory Press, 1989) are well known to those of skill in the art as are other suitable hybridization buffers. Thus, for example, an alternate set of conditions suitable to accomplish low stringency hybridization comprise a temperature of about 37°C, a formamide concentration of about 20%, and a salt concentration of about 5X SSC.

As used herein, the term "degenerate" refers to codons that differ in at least one nucleotide from a reference nucleic acid, e.g., SEQ ID NO:l, but encode the same amino acids as the reference nucleic acid. For example, codons specified by the triplets "UCU", "UCC" , "UCA", and "UCG" are degenerate with respect to each other since all four of these codons encode the amino acid serine.

As used herein, a nucleic acid "probe" is single-stranded DNA or RNA, or analogs thereof, that has a sequence of nucleotides that includes at least 14, preferably at least 20, more preferably at least 50, contiguous bases that are the same as (or the complement of) any 14 or more contiguous bases set forth in any of SEQ ID NO:l or SEQ ID NO:3. In addition, the entire cDNA encoding region of an invention protein may be used as a probe. Probes may be labeled by methods well-known in the art, as described hereinafter, and used in various diagnostic kits.

As used herein, the terms "label" and "labeling means" refer to single atoms and molecules that are either directly or indirectly involved in the production of a detectable signal. Any label or labeling means can be linked to invention nucleic acid probes, expressed proteins, polypeptide fragments, or antibody molecules. These atoms or molecules can be used alone or in conjunction with additional reagents. Such labels are themselves well-known in clinical diagnostic chemistry.

The labeling means can be a fluorescent labeling agent that chemically binds to antibodies or antigens without denaturation to form a fluorochrome (dye) that is a useful immunofluorescent tracer. Suitable fluorescent labeling agents are fluorochromes such as fluorescein isocyanate (FIC) , fluorescein isothiocyanate (FITC) , 5-dimethylamine-l-naphthalenesulfonyl chloride (DANSC) , tetramethylrhodamine isothiocyanate (TRITC) , lissamine, rhodamine 8200 sulfonyl chloride (RB-200-SC) , and the like. A description of immunofluorescent analytic techniques is found in DeLuca, "Immunofluorescence Analysis", in Antibody As a Tool , Marchalonis et al., eds., John Wiley & Sons, Ltd. , pp. 189-231 (1982) , which is incorporated herein by reference.

The labeling means can also be an enzyme, such as horseradish peroxidase (HRP) , glucose oxidase, and the like. In such cases where the principal indicating group is an enzyme, additional reagents are required for the production of a detectable signal. Such additional reagents for HRP include hydrogen peroxide and an oxidation dye precursor such as diaminobenzidine. An additional reagent useful with glucose oxidase is 2,2'-azino-di-(3-ethyl-benzthiazoline-G-sulfonic acid) (ABTS) .

Radioactive elements are also commonly employed as labeling agents. An exemplary radiolabeling agent is a radioactive element that produces gamma ray emissions. Elements which emit gamma rays, such as I, I, I, I and Cr, represent one class of radioactive element indicating groups. Particularly preferred is I. Another group of useful labeling means are those elements such as

C, F, O and N which emit positrons. The positrons so emitted produce gamma rays upon encounters with electrons present in the animal's body. Also useful is a beta emitter, such as P, indium or H.

The linking of a label to a substrate, i.e., labeling of nucleic acid probes, antibodies, polypeptides, and proteins, is well known in the art. For instance, an invention antibody can be labeled by metabolic incorporation of radiolabeled amino acids provided in the culture medium. See, for example, Galfre et al., Meth . Enzymol . 73:3-46 (1981). Conventional means of protein conjugation or coupling by activated functional groups are particularly applicable. See, for example, Aurameas et al., Scand. J. Immunol . Vol. 8, Suppl. 7:7-23 (1978), Rodwell et al., Biotech . 3:889-894 (1984) and U.S. Patent No. 4,493,795.

The present invention will now be described in greater detail with reference to the following non-limiting examples:

Example 1 Construction of cDNA Library

WEHI-7TG cells were used as the source of RNA for the construction of a cDNA library enriched in sequences induced by glucocorticoids. WEHI-7TG is a thioguanine-resistant derivative of the glucocorticoid-sensitive BALB/c murine thymoma line WEHI-7 (Bourgeois and Newby, Cell 11:423-430 (1977); Harris et al., J . Immunol . 110:431-438 (1973)). Cells are grown in Dulbecco modified Eagle medium supplemented with 10% fetal calf serum (GIBCO Laboratories, Grand Island, N.Y.). WEHI-7TG cells were treated for 5 hours with 1 mM tria cinolone acetonide and 12 mM forskolin (an agent which increases intracellular levels of cAMP) . Total cellular RNA was extracted from cells and BALB/c mouse tissues according to the single step method of RNA isolation by acid guanidinium thiocyanate -phenol- chloroform extraction described by Chomczynski and Sacchi (Anal. Biochem 162:156-159 (1987)). Poly (A)⁺ RNA was isolated by affinity chromatography on oligo (dT) cellulose by standard procedures. Double-stranded cDNA was synthesized from poly (A)⁺ RNA using the Amersham cDNA synthesis kit (Amersha Corp.) The cDNA was methylated prior to ligation with EcoRI linkers to protect internal EcoRI sites. After removal of excess linkers, a portion of the cDNA was inserted into ΛZAP vectors (Stratagene) to produce a library of 5 x 10 independent recombinants. The unamplified library was plated at 50,000 pfu (plaque forming units) per 150 mm dish in 0.7% top agarose supplemented with 30% glycerol, and filter lifts were prepared with Hybond-N (Amersham Corp.). The plates were stored at -70°C.

Example 2

Synthesis of cDNA Substraction Hybridization Probe

Single-stranded [ 32P] cDNA was prepared using poly (A)^* RNA isolated from WEHI-7TG cells treated with triamcinolone acetonide and forskolin as described above. Using the Amersham cDNA synthesis kit, the first strand synthesis reaction mixture was modified to contain 0.5 mM each of dATP, dGTP, and dTTP, 0.1 mM dCTP, 50 mg of actinomycin D per ml, and 200 mCi of [α- P] dCTP (3,889 Ci/mmol) . After first-strand synthesis, the template RNA was removed by incubation in 0.2 N NaOH at 70°C for 20 minutes. The reaction was neutralized with HC1, and the cDNA was ethanol precipitated in the presence of 2.5 M ammonium acetate. The specific activity of the cDNA was approximately 1 x 10 8 to 2 x 108 cpm/mg. The [32P] cDNA was hybridized to a 40-fold mass excess of poly (A)^* RNA from uninduced CXG56D3 cells. CXG56D3 is a variant of WEHI-7TG resistant to both glucocorticoids and cAMP. Hybridization was performed at a R₀t of 1,000 (mol/liter) x sec. in 0.5 M sodium phosphate buffer (pH 7), 0.1% sodium dodecyl sulfate (SDS) and 1 mM EDTA (ethylenediaminetetraacetic acid) .

Unhybridized cDNA was collected by hydroxylapatite chromatography (Bio-Rad) in 0.12 M phosphate buffer-0.1%

SDS at 60°C. After two cycles of hybridization, the remaining single-stranded, [ P]-labeled cDNA was collected and used for library screening as described below. Example 3 Library Screening

Library filters prepared as described in Example 1 were hybridized at 42°C in a solution containing the radiolabeled cDNA subtraction probe (prepared as described in Example 2) in 50% formamide, 5 x SSPE (1 x SSPE is 0.18 M NaCl, 0.01 M NaHP0₄, 1 mM EDTA), 0.5% SDS, 5% (wt/vol) dextran sulfate, 7 x Denhardt solution (1 x Denhardt solution is 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin) , and 200 mg of denatured salmon sperm DNA per ml. Filters are washed in 0.3% SSPE-0.1% SDS at 65°C and then exposed to Kodak XAR-5 film at -70°C. Approximately 1,000 plaques per 5 x 10 representatives gave a positive signal. Randomly chosen positive plaques (100) were rescreened at low density by differential hybridization utilizing duplicate lifts hybridized to cDNA probes prepared from poly (A)^* RNA from WEHI-7TG cells treated with triamcinolone acetonide and forskolin for 5 hours or from poly(A)^* RNA from untreated CXG56D3 cells. Plaques (i.e., cDNA clones) giving hybridization signals of different intensities with the two probes were selected for further study.

Example 4 Characterization of Positive Clones

Northern blot analysis was used to verify tha the selected cDNA clones represented genes induced b glucocorticoids and/or forskolin in the WEHI-7TG cell line. cDNA inserts from each ZAP plaque were subcloned int pBluescript(SK-) plasmid vector using an in vivo excisio protocol provided by the manufacturer (Stratagene) . Plasmid vector DNA representing all cDNA inserts wa digested with EcoRI and purified by agarose ge electrophoresis. Radioactive [ 32P]-labeled probe (1 x 10 to 2 x 10⁹ cpm/mg) was prepared from each cDNA insert using the Mulitprime DNA labeling kit (Amersham Corp.).

Total cellular RNA was prepared from untreated CXG56D3 cells, untreated WEHI-7TG cells, WEHI-7TG cells treated for 5 hours with either dexamethasone (1 mM) , forskolin (12 mM) , or both drugs in combination, or from BALB/c mouse tissues (brain, heart, kidney, liver, lung, spleen, and thymus) according to the procedure described in Example 1. The RNA (10 mg) was fractionated in 1% agarose gel containing 2.2 M formaldehyde and transferred to Hybond-N. The resulting filters were hybridized with radioactive [ P] labeled probe prepared from each insert. Hybridization was carried out at 42°C in 50% formamide, 0.5 M sodium phosphate buffer (pH 7.2), 1 mM EDTA, 1% bovine serum albumin and 5% SDS. Filters were washed in 0.3 x SSPE-0.1% SDS at 65°C and exposed to Kodak XAR film at -70°C. cDNA clones were identified that represent 13 different glucocorticoid- and cAMP- regulated genes in the WEHI-7TG cell line (Harrigan et al., Mol . Cell . Biol . 9:3438-3446 (1989); Baughman et al., Mol . Endocrinol . 5:637-644 (1991)). One of these cDNA clones, designated clone 213, represents an approximately 3.5 to 4 kb mRNA that is induced by glucocorticoids and slightly repressed by forskolin in the WEHI-7TG cell line and expressed predominantly in thymus tissue.

Example 5 Nucleotide Sequence Analysis

Clone 213 contained a cDNA insert of approximately 2.2 kb. Sequence determination and analysis was performed by the dideoxy chain termination method (Sanger et al., Proc . Natl . Acad. Sci . USA 74:5463-5467 (1977)) using the Sequenase DNA sequencing kit (US Biochemicals) and commercial primers for the pBluescript (SK-) vector. Preliminary sequence data was obtained by sequencing one strand from each end of the cDNA insert to determine if the sequence gene is a new or previously described gene. The presence of a poly(A) tail confirmed that the cDNA insert originated from the 3' portion of the corresponding mRNA. A computer search was run to screen for sequences matching sequences represented by a minimum of 230 nucleotides from each end of the vector junction. The GenBank (version 65) nucleotide data base was searched using the FASTA program of Pearson and Lip an (Proc. Natl . Acad . Sci . USA 85:2444-2448 (1988). Search results failed to identify any sequences having significant similarity.

An approximately 500 base pair fragment from the 5' end of the 2.2 kb cDNA insert of clone 213 was isolated by restriction enzyme digestion with EcoRI and Xhol, gel purified and radiolabeled with P to provide a hybridization probe for screening for full-length cDNAs. A murine thymus ΛZAP cDNA library (Stratagene) was screened with this probe. Hybridization was carried out at 42°C under similar conditions to those described in Example 3 with the exception that dextran sulfate was not added to the hybridization solution. A cDNA clone of approximately 3.5kb was isolated. In order to determine additional sequence information from potentially longer clones, an approximately 600 base pair fragment from the 5' end of the 3.5kb clone was isolated by restriction enzyme digestion with Sad, gel purified, radiolabeled with P, and used as a hybridization probe to rescreen the same library.

A cDNA clone, designated clone 213-12A, was isolated upon rescreening the murine thymus ΛZAP cDNA library. The nucleotide sequence of approximately 2.3 kb of this clone (region 5' to an internal Xhol restriction site) was determined using Erase-A-Base (Promega Corporation) nested deletions, and the Sequenase Version 2 sequencing kit (US Biochemicals) . The nucleotide sequence of clone 213-12A, contains 2226 nucleotides (see SEQ ID NO:l) .

Example 6 Amino Acid Sequence

Computer analysis of clone 213 reveals the presence of an open reading frame (ORF) encoded by nucleotides 161 - 1528, as shown in SEQ ID NO:l. Analysis of this open reading frame reveals a protein of 456 amino acid residues with a molecular weight of about 50,969 Da and a pKi of 7.8. The amino acid sequence, from amino to carboxyl terminus, is shown in SEQ ID NO:2. Searches of the SwisProt data base using the FASTA program reveal that the deduced protein is a member of the family of proteins (FKBPs) that bind immunosuppressive drugs FK-506 and rapamycin.

Example 7 Production of protein

Uncapped RNA corresponding to the cDNA insert of clone 213-12A was synthesized in vitro using Riboprobe transcription system (Promega Corporation) and the pBluescript(SK-) vector-encoded T3 or T7 transcription initiation control regions. The RNA was incubated in a rabbit reticulocyte lysate (Promega Corporation) in the presence of 7.5 mCi [ ⁵S] methionine (1053 Ci/mmol, Amersham Corp.) and the translation products were visualized by separation on 10% SDS-polyacrylamide gels followed by treatment with Fluorohance (Research Products International) , drying, and autoradiography. A protein product corresponding to the open reading frame of clone 213-12A was detected. The protein migrated at an apparent molecular weight of approximately 51 kDa. A cDNA corresponding to the open reading frame of clone 213-12A was expressed in E. coli using the pGEM-3X (Pharmacia) vector that produces proteins in E . coli with an amino terminal glutathione S-transferase (GST) tag that allows purification of the recombinant protein by glutathione sepharose affinity chromatography. The pGEM-3X vector also contains a protease factor Xa cleavage recognition site which permits removal of the glutathione S-transferase tag from the recombinant protein after purification.

cDNA corresponding to the open reading frame of clone 213-12A was amplified by PCR technology using suitable primers that include restriction digestion sites for subcloning into the pGEM-3X vector. The forward primer [CGGGATCCGGACAATGACTACTGATGAG, 28-mer; SEQ ID NO:8] contains a BamHI restriction site 5' to 5 nucleotides which precede (1) the translational initiation site (ATG) of the open reading frame, and (2) the sequence corresponding to the first 4 amino acids of the protein. The reverse primer rCGGAATTCCGTCATACATGGCCTTTGGC. 28-mer; SEQ ID NO:9] contains an EcoRI restriction site 5' to 2 nucleotides which precede (1) the sequence complementary to the stop codon for translation (TCA) and (2) the last _^5 nucleotides of the open reading frame. The cDNA was amplified using these primers in a thermocycler (EriComp) under the following cycle conditions: 5 min initial denaturation at 94°C, followed by 35 cycles of 30 sec primer annealing at 60°C, 1 minute extension at 72°C, and 30 sec. denaturation at 94°C, followed by a final extension at 72°C for 10 minutes.

The amplified product was purified by agarose gel electrophoresis, digested with EcoRI and BamHI, repurified by electrophoresis and cloned into EcoRI-BamHI digested pGEM-3X vector DNA. The resultant recombinant plasmid was transformed into BL21 E . coli cells and selected by ampicillin resistance. An ampicillin-resistant transformed colony was inoculated into 500 ml Terrific Broth (Gibco BRL) containing 100 mg ampicillin per ml and grown with aeration at 37°C until the culture reached an optical density at 600 nm of 0.5. Protein expression was induced by the addition of 0.5 M IPTG (isopropyl-b-D-thiogalactopyranoside) . After 2 hours the cells were harvested by centrifugation (4,000 g, 15 minutes, 4°C) . Subsequent steps in the protein purification, described below, were performed on ice to minimize protein degradation.

The resulting cell pellet was washed with 1 x PBS containing 5 mM MgCl₂, repelleted as described above and then resuspended in 20 ml lysis buffer containing 50 mM Tris HC1 (pH 7.9), 100 mM KCl, 1% Triton X-100, 12.5 mM MgCl₂, 10 mM dithiothreitol (DTT) , 0.1 mM phenylmethylsulfonylfluoride (PMSF) , 2 mg/ml benzamidine, 1 mg/ml pepstatin A, 4 mg/ml leupeptin, lOmg/ml aprotonin, and 20 mg/ml soybean trypsin inhibitor. The cells were lysed by 3 cycles of freeze-thawing in dry ice, followed by clearing of the lysate by centrifugation (100,000g, 30 minutes, 4°C) .

Recombinant protein in the lysate was bound to Glutathione Sepharose 4B (Pharmacia, 2 ml beads per 500 ml culture) by batch incubation at 4°C for 2 hours with rotation. Sepharose beads containing the bound protein were collected by centrifugation (400g, 5 minutes, 4°C) and washed at 4°C with 3 volumes of wash buffer per ml of beads, as follows: Wash I: 1 x PBS, 0.1% Triton X-100, 2 mM DTT,

0.1 mM PMSF; Wash II: 0.3 M NaCl, 50 mM Tris HCl (pH 7.9), 2 mM DTT, 0.1 mM PMSF; and Wash III: same as Wash I. The beads were collected between washes by centrifugation (400 g, 5 minutes, 4°C) . Purified recombinant protein was eluted from the beads by incubation for 5 minutes at 4°C in 50 mM Tris HC1 (pH 7.9), 10 mM glutathione, 2 mM DTT, and 0.1 mM PMSF.

Alternatively, after wash III, a fourth wash was performed in factor Xa cleavage buffer (Tris HC1 (pH 8.0), 100 mM NaCl, 1 mM CaCl₂) to remove residual PMSF, followed by incubation of the beads in factor Xa cleavage buffer containing 1% mass ratio of factor Xa (Prozyme Inc.) to recombinant protein for 2 hours at room temperature, with rotation. This alternate procedure resulted in recombinant protein containing 4 non-native amino acids at the amino terminus. Both protein products were analyzed on SDS-polyacrylamide gel electrophoresis. The recombinant protein and the factor Xa cleaved protein migrated at apparent molecular weights of 79 and 51 kDa, respectively.

Example 8 Antibody Preparation

Polyclonal rabbit antisera specific for protein encoded by cDNA corresponding to the open reading frame of clone 213-12A was prepared against a synthetic peptide coupled to KLH (Pierce Chemical Company) . The synthetic peptide had the amino acid sequence YGESQAMEEGKAKGHV (SEQ ID NO:10), which corresponds to the 14 carboxyl terminal amino acids residues of the amino acid sequence shown in SEQ ID NO:2, plus two residues (YG) important for coupling. The peptide was synthesized according to manufacturer's protocol on the Synergy peptide synthesizer (Applied Biosystems) . Peptide and KLH carrier were coupled in a 1:3 weight ratio using bisdiazotized benzidine (BDB) as a coupling agent (Vaughn et al., Meth . Enzymol . 168:588-617

(1989)). BDB reagent was removed following coupling by dialysis against 0.9% NaCl. New Zealand white rabbits were injected subcutaneously at 3 week intervals with 0.5 mg (injections 1,2, and 3) or 0.25 mg (injections 4 and 5) coupled peptide in Freunds complete (injection 1) or incomplete (injections 2-5) adjuvant. Bleeds were taken 2 weeks post injection, the blood allowed to clot, and the serum collected by centrifugation (500g, 10 minutes) . The antisera were used in Western analyses to detect the recombinant immunophilin protein produced in E . coli cells and the endogenous protein produced in WEHI-7TG cells and thymus tissue, which protein corresponds to the amino acid sequence shown in SEQ ID NO:2.

E. coli cells harboring the recombinant plasmid induced by IPTG as described above, as well as WEHI-7TG cells, were collected by centrifugation (500g, 10 minutes) and lysed in 1 x SDS sample buffer (Laemmli, U.K., Nature 227:680-685 (1970)). Murine tissues (brain, heart, kidney, liver, lung, spleen and thymus) were excised, frozen in liquid nitrogen, and disrupted by homogenation in a lysis buffer containing 25 mM Tris HCl (pH 7.5), 5 mM DTT, 50 mM NaF, 2 mM PMSF, and 0.015% Triton X-100 using a Polytron (Brinkman Instruments) . Tissue extracts were clarified by centrifugation (20,000g, 30 minutes, 4°C) . Protein concentrations were determined by the Coomassie Blue dye binding assay (Bio-Rad protein assay) . Extract proteins (50 μg) were heated in 1 x SDS sample buffer for 2 minutes at 90°C followed by separation on a 10% SDS-PAGE. The resolved proteins were transferred to nitrocellulose (BA85, Schleicher and Schuell) using a semidry transfer apparatus (LKB) in 1 x SDS gel running buffer containing 20% methanol at 1mA per cm for 1 hour. The filters were blocked in 1 x PBS containing 5% powdered milk overnight at 4°C, followed by incubation with 213-peptide specific antisera (1:500 dilution) in 1 x PBS containing 1% powdered milk for 2 hours at room temperature. After washing with 1 x PBS containing 0.05% Tween-20, the filters were incubated with alkaline phosphatase conjugated goat anti-rabbit IgG (BioRad) at a 1:7500 dilution for 1 hour at room temperature in 1 x PBS containing 1% powdered milk. The filters were washed, first with 1 x PBS containing 0.05% Tween-20 and then with 1 x PBS alone. The immobilized antibody complexes were detected by a colori etric assay using the NBT/BCIP color development reagent (BioRad) . The peptide antisera prepared in this Example specifically recognize the recombinant immunophilin protein produced in E . coli, and the invention protein present in WEHI-7TG cells and predominant in thymus tissue.

Example 9 Human cDNA Library Screening

The Clontech human thymus 5'-stretch λgtll cDNA library (Catalog #HL1074b) was used as a source of human cDNA clones corresponding to the mouse immunophilin protein encoded by clone 213-12A. The library (2.25 x 10⁶ representatives) was plated at 75,000 pfu (plaque forming units) per 150 mm dish according to the instructions provided by the manufacturer and filter lifts were prepared with Hybond-N (Amersham Corp.). An 870 base pair fragment corresponding to nucleotides 463-1333 as shown in SEQ ID NO:l was isolated by Bglll restriction enzyme digestion, gel purified and radiolabeled with [³²P] dATP to 1-2 x 10⁹ cpm/μg using the Multiprime DNA labeling kit (Amersham Corp.) . Hybridization to the above filters was carried out at 42°C in 50% formamide, 0.25 M NaHP0₄ (pH 7.2), 0.25 M NaCl, 1 mM EDTA, 7% SDS, and 100 μg/ml denatured salmon sperm DNA for 20 hours. Filters were washed in 2 x SSC (1 x SSC is 0.15 M NaCl, 0.05 M sodium citrate), 0.1% SDS at 52°C, followed by washing in 25 mM NaHP0₄ (pH 7.2), 1 mM EDTA, 0.1% SDS at 52°C. The filters were exposed to Kodak XAR film at -70°C. Positive clones (129 candidates) were identified and subjected to a second round of screening. A DNA fragment corresponding to nucleotides 1-561 as shown in SEQ ID NO:l was isolated by Sacl restriction enzyme digestion, purified, and radiolabeled as described above. Hybridization and detection of positive clones was performed as above, resulting in the identification of 39 positive clones that hybridize to both of the radiolabeled probes.

Example 10 Nucleotide Sequence Analysis of Positive Human Clones

Human cDNA inserts were isolated from purified plaques giving a positive hybridization signal using protocols designed to obtain DNA from lambda lysates as described by the manufacturer (Clontech) . Thus, Y1090r^' bacterial host cells were incubated at 37°C overnight in 15 ml of LB broth (pH 7.5) containing 10 mM MgS0₄ and 0.2% maltose. The cells were then pelleted, and resuspended in 10 mM MgS0₄, then stored at 4°C for a maximum of 2-3 days before use.

For phage stock preparation, the ratio of phage to bacterial host must be empirically determined. Typically, a yield of 100-200 μg of phage DNA can be expected from 500 ml of phage lysate. An appropriate number of lytic phage (typically 600-800 plaques per 90-mm plate) are plated in the presence of 200 μl of host cells, so that a single plaque can be easily removed. The plates are then incubated overnight at 37°C. An agar plug containing a single plaque is transferred to a microfuge tube containing 200 μl of IX lambda dilution buffer. Transfer is accomplished, for example, with the end of a pasteur tube. A drop of chloroform is then added, and the tube vortexed briefly. Phage are then eluted at 4°C overnight (or at 37°C with shaking for 4-6 hr. The microfuge tube is then spun at 10,000 rpm (i.e., -8,000 xg) for ~2 min to remove debris. The supernatant is then titered, and plated in the presence of 600 μl of host cells, on a 150-mm LB agarose plate containing 10 mM MgS0₄. Based on the titer values obtained, enough phage are plated so that near confluency is obtained in 5-7 hr. The plates are then incubated at 37°C for 5-7 hr. Ten ml of IX lambda dilution buffer is then added to the plate, and incubated at 4°C overnight. A few drops of chloroform are then added to the plate, which is swirled briefly. The liquid is then poured from the plate into a sterile 50 ml polypropylene tube. Two ml of chloroform are then added to the plate lysate, which is then vortexed for ~2 min. Sample is then centrifuged at -7,200 xg for -10 min. The high titer stock ssuuppeerrnnaattaanntt ((wwhhiicchh sshhoouulldd hhaa^ve a titer of about 10 pfu/ml) is then collected and saved.

Lysate dilutions are then prepared from the high titer stock such that approximately 10 pfu/ml are obtained on each 150-mm LB agarose plate containing 10 mM MgS0₄. A single, isolated colony is picked from an E. coli Y1090r^" host cell plate and used to inoculate LB broth containing 10 mM MgS0₄ and 0.2% maltose. This is then incubated on the shaker at -200 rpm at 37°C overnight. The appropriate number of tubes are then set up with 600 μl of bacterial culture and diluted lysate. This is then incubated in a 37°C water bath for -15 min. 5.5-6.5 ml of melted LB soft top agarose + 10 mM MGS0₄ are added. Inoculum is then mixed and poured onto 150-mm plates. The plates are swirled quickly while pouring to allow even spreading of the agarose. The plates are then incubated at 37°C for 5-6 hr.

12 ml of IX lambda dilution buffer is added to each plate, and the plates stored at 4°C overnight. The plates are then incubated at room temperature for 1 hr with constant shaking. The IX lambda dilution buffer solution is removed and saved; and the plate surface is rinsed with an additional 2 ml of IX lambda dilution buffer. The lambda dilution buffer solutions are pooled. This is the plate lysate.

For the preparation of large-scale liquid lysate, 1-3 ml of phage stock (1-3 x 10 pfu) are added to 1 L of host cells grown in LB broth to an OD₅₀₀ of 0.6. The culture is shaken at 37°C in a 4-L flask until lysis is apparent (6-10 hr, depending upon the vector) . After lysis, 10 ml of chloroform are added, and the flask shaken for an additional 15 min. The flask is removed from the shaker. The lysate is centrifuged at -7,200 xg for -10 min at 4°C in polypropylene bottles. The supernatants are combined, and titer checked. This is the liquid lysate which is now ready for DNA extraction.

0

If the titer is below -10 pfu/ml, the above steps should be repeated. For large quantities of DNA, the titer must be greater than -10 pfu/L. Standard yield = 440 μg of DNA/L: (10¹³ pfu/L) x (40 kb/pfu) x (660,000 g/6 x 10²³ kb) = 440 μg Of DNA/L.

If the titer is insufficient, the ratio of phage to bacteria should be increased. If lysis appears complete within 2-3 hr, the ratio of phage to bacteria is too high, and the bacterial population has been lysed prematurely. If lysis occurs too quickly, the above steps should be repeated.

For lysate processing, the bottled, pooled lambda dilution buffer solutions (obtained either from plate or liquid lysates) are centrifuged at -10,000 xg for 10 min in order to pellet debris. DNase 1 is added to the supernatant to 1 μg/ml and RNase A is added to 5 μg/ml. The resulting mixture is incubated at room temperature for 30 min. 100% chloroform is then added to a final concentration of 5%, and the solution vortexed for 30 sec. Sample is then centrifuged at -10,000 xg for 10 min at 4°C to pellet the debris.

The aqueous phase is transfered to a new centrifuge tube. An equal volume of 20% PEG/2.0 M NaCl is added to the aqueous phase, which is incubated on ice for at least 1 hr. Sample is then centrifuged at -10,000 xg for 10 min at 4°C to pellet the phage. The precipitated phage are then centrifuged at -10,000 xg for 15 min at 4°C, and the supernatant discarded. A grayish smear should be evident on the side of each bottle.

It is preferable to remove as much of the PEG solution as possible as PEG can inhibit restriction enzymes.

The pellets are resuspended in 32 ml of IX lambda dilution buffer. The phage suspension is then transfered into two 50-ml polypropylene tubes, and an equal volume of chloroform added. The resulting mixture is then vortexed for 30 sec, then centrifuged at -7,200 xg for 10 min. The supernatant is collected, being careful to leave the PEG interface behind.

0.5 g of CsCl are added per ml of phage suspension, which is then poured into a 40-ml UltraClear™ tube, and centrifuged at -90,000 xg for 2 hr at 20°C. The supernatant is poured off, and the clear, sticky phage pellet resuspended in 1 ml of IX lambda dilution buffer. The resulting mixture is then transfered to a 1-ml microcentrifuge tube, and debris spun down at 15,000 rpm for 10 min.

For DNA extraction, EDTA is added to the phage DNA to 20 mM, SDS to 0.5%, and proteinase K to 50 μg/ml final concentrations. The resulting mixture is then incubated at 65°C for 1 hr. An equal volume of phenol:chloroform is added, and the solution mixed by gentle inversion for 10 min. The resulting mixture is then centrifuged at 7,000 rpm for 10 min at room temperature (or for 5 min in a microcentrifuge for smaller volumes) . The supernatant is collected, and the above steps are repeated until the interface is clean (usually one extraction is suf icient) .

The above steps are repeated with chloroform only to remove any residual phenol (the small amount of aqueous material left at the interface can be collected separately in 1.5-ml microcentrifuge tubes, centrifuged, and combined with the larger fraction). 1/10 volume of 3M NAOAc and 2.5 volumes of 95% ethanol are added, and the sample stored at -20°C for at least 1 hr for DNA precipitation. DNA is then centrifuged at 12,000-15,000 rpm for 15 min at room temperature. The supernatant is then poured off, and the pellet washed with 70% ethanol. Sample is then centrifuged for 5 min, and the supernatant poured off. The residual ethanol is spun down—the last few drops can be removed with a micropipette.

The pellet is allowed to dry until the edges of the pellet begin to turn clear. If the DNA is not completely dry, it will resuspend well in TE buffer. If the DNA is dry, 1 ml of TE buffer should be added, and the pellet allowed to resuspend at 4°C overnight.

The DNA should form a sharp band on an agarose gel with some contaminating RNA. RNase A can be added along with restriction enzymes, thus eliminating an additional digestion. A spectrophotometer reading should give characteristic curves, with a maximum absorption at 260 nm. The A_260/280 ratio should be >1.8.

For insert excision, 5-10 μl of purified DNA are digested with EcoRI at 37°C for 3-16 hr. Samples are 32 heated at 70°C for 10 min to inactivate the enzyme. The digest is then analyzed on a 0.8% agarose minigel. Depending on lysate titer, digestion of the entire isolate may be necessary in order to view the digest pattern. Precipitation of more than the usual volume of lysate can be performed (i.e., >10 ml) if a low titer is anticipated.

Human cDNA inserts were ligated to EcoRI-digested pBluescript (SK-) vector DNA, transformed into XL-1 blue

E . coli host cells, selected by ampicillin resistance, and positive clones identified by restriction enzyme analysis.

Clone Hm51-35 was identified to contain an approximately 2.3 kb human cDNA insert. The nucleotide sequence of approximately 1619 base pairs of this clone was determined by automated sequence analysis (Applied Biosystems) using a succession of synthetic primers employing well known gene-walking methodology.

Example 11 Amino Acid Sequence of Human Clone

Computer analysis of clone Hm51-35 reveals the presence of an open reading frame (ORF) encoded b nucleotides 154-1524 as shown in SEQ ID NO:3. Analysis of this open reading frame reveal a protein of 457 amino aci residues with a molecular weight of about 51,221 Da and pKi of 5.65. The amino acid sequence, from amino t carboxyl terminus, is shown in SEQ ID NO:4.

Example 12 Peptidylprolyl cis-trans Isomerase Assay

The peptidyl-propyl isomerization activity o recombinant invention immunophilin protein is measure using an assay described by Fischer et al.. Natur

337:476-478 (1989). The Fischer assay measures th immunophilin catalyzed cis-trans isomerization of a N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (SEQ ID NO:6) substrate by coupling the reaction to the specific cleavage of the trans isoform of the substrate and release of p-nitroanilide by chymotrypsin. In this assay, N-succinyl-Ala-Leu-Pro-Phe-p-nitroanilide (Bachem; SEQ ID NO:7) is employed as the substrate at a final concentration of 72 μK, and chymotrypsin is used at a concentration of 6 μVL. Release of p-nitroanilide by chymotrypsin is quantified by measuring the increase in absorbance at 405 nm by spectroscopy. Protein concentrations of stock solutions of the recombinant invention immunophilin protein are determined by the Coomassie Blue dye binding assay (BioRad). Increasing concentrations (0.5-5 mg/ml) of the protein are added to a reaction mixture (1.5 ml) containing substrate 0.1 M Tris-HCl (pH 7.8). The mixture is incubated at 15°C for 15 minutes, followed by addition of chymotrypsin. The generated data are fitted to a simple first-order rate equation.

Inhibition of PPIase activity is performed in the presence of increasing concentrations (l-iooo nM) of the immunosuppressant drugs, i.e. rapamycin and FK-520. Assays are performed as described above except that the amount of recombinant protein is kept constant (7.5 mg) . The recombinant invention immunophilin protein exhibits PPIase activity which is inhibited by immunosuppressive drugs such as rapamycin and FK-506 like compounds.

Example 13 Binding Assay

Binding and displacement of [ H] dihydro FK-506 from invention immunophilin protein is measured by a modification of the LH-20 method (Handschumacher et al., Science 226:544-547 (1984)). Reaction mixtures (100 μl) containing at least 1 nM invention immunophilin protein and 34

100 nM [³H] dihydro FK-506 (New England Nuclear) are incubated for 30 minutes in TSK buffer (20 mM sodium phosphate (pH 6.8), 50 M Na₂S0₄,5 mM β-mercaptoethanol, and 1 mM EDTA containing 0.5% (w/v) bovine serum albumin. Following incubation, the reaction mixture is divided into two equal 50 μl portions, A and B. Bound and unbound [³H]dihydro FK-506 are separated from portion A by chromatography on a 1.5 ml Sephadex LH-20 column (Pharmacia) . The material which elutes with TSK buffer in this portion represents bound drug. Radioactivity in the unfractionated portion B (representing total bound and free) and the eluted material from portion A are quantified by liquid scintillation counting to obtain values of relative drug binding. Drug and protein concentration dependent binding of [ H] dihydro FK-506 to invention immunophilin is observed in this assay. Rapamycin

(Wyeth-Amerst Research Co.) addition (50-500 μM) to the above-described reaction mixture competitively inhibits [ H] dihydro FK-506 binding to invention immunophilin.

Example 14

Calcineurin Phosphatase Inhibition Assay

The phosphatase activity of calcineurin, and the subsequent inhibition of this activity by immunophilin-drug complexes, is determined in a modified assay described by Liu et al., Cell 66:807-815 (1991) and Manalan and Klee, Proc. Natl . Acac . Sci . USA 80:4291-4295 (1983) in which a phosphorylated peptide fragment (RII) from the regulatory subunit of cAMP-dependent kinase acts as substrate. Reaction mixtures (60//1) containing 40 mM Tris-HCl (pH 7.5), 100 mM NaCl, 6 mM MgCl₂, 0.1 mM CaCl₂, 0.1 mg/ml BSA, 0.5 mM DTT, 0.1 μg bovine brain calmodulin (Sigma Chemical Company) , 0.05 units bovine brain calcineurin, 30 μM FK-520 and increasing concentrations of recombinant invention immunophilin protein (0.1 nM to 10 μl ) are incubated at 30°C for 30 minutes prior to addition of phosphorylated substrate peptide. Reactions are initiated by addition of the peptide (40 μM [ P] RII peptide (432 cpm/pmol) , and the dephosphorylation reaction is allowed to proceed for 10 minutes at 30°C. Reactions are terminated by the addition of 0.5 ml of 5% trichloroacetic acid containing 100 mM sodium phosphate (stop buffer) and applied to 0.5 ml Dowex AG 50W-X8,H^* column. Free [ P] phosphate is eluted from the column with 0.5 ml of stop buffer and quantified by liquid scintillation counting.

While the invention has been described in detail with reference to certain preferred embodiments thereof, it will be understood that modifications and variations are within the spirit and scope of that which is described and claimed.

SUMMARY OF SEQUENCES

SEQ ID NO:l is the nucleic acid sequence and the amino acid sequence of cDNA encoding a murine-derived immunophilin protein of the present invention.

SEQ ID NO:2 is the amino acid sequence of an immunophilin protein of the present invention encoded by SEQ ID NO:l.

SEQ ID NO:3 is the nucleic acid sequence and the amino acid sequence of cDNA encoding an human-derived immunophilin protein of the present invention.

SEQ ID NO: 4 is the amino acid sequence of the immunophilin protein of the present invention encoded by SEQ ID NO:3.

SEQ ID NOs:5, 6 and 7 are substrates for immunophilin catalyzed cis-trans isomerization reactions.

SEQ ID NOS:8 and 9 are forward and reverse PCR primers, respectively, for amplification of the open reading frame of clone 213-12A.

SEQ ID NO:10 is the amino acid sequence of a synthetic peptide used for the preparation of polyclonal rabbit antisera specific for protein encoded by clone 213-12A. SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Bourgeois-Cohn Ph.D, Suzanne H. Baughman Ph.D., Gail A.

(ii) TITLE OF INVENTION: Novel Immunophilins and Corresponding Nucleic Acids

(iii) NUMBER OF SEQUENCES: 10

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Pretty, Schroeder, Brueggemann & Clark

(B) STREET: 444 South Flower Street, Suite 2000

(C) CITY: Los Angeles

(D) STATE: CA

(E) COUNTRY: USA

(F) ZIP: 90071

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.25

(vi) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 08/323,367

(B) FILING DATE: 14-OCT-1994

(C) CLASSIFICATION:

(vii) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: US

(B) FILING DATE:

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Stephen E. Reiter

(B) REGISTRATION NUMBER: 31,912

(C) REFERENCE/DOCKET NUMBER: P41 9931

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (619) 546-4737

(B) TELEFAX: (619) 546-9392

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2226 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: unknown

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic) (vii) IMMEDIATE SOURCE:

(B) CLONE: 213-12A

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 161..1528

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

CTCCGGCGGC GGTGTCCGGC GCGCGGCTGA GCGACCGAGC GTGCGGACGG AGCGGCGGCC 60

TGCTGGGCGG GCTGAGCGGC GCGCGCGGCG GCGGAGAGAC GCGGAGCGAG GGACGCGGCG 120

GCGGCGGACG CGGCGACAGG TCTTCTACTT ACAAAGGACA ATG ACT ACT GAT GAG 175

Met Thr Thr Asp Glu 1 5

GGC ACC AGT AAC AAT GGA GAG AAC CCA GCA GCC ACC ATG ACT GAG CAG 223 Gly Thr Ser Asn Asn Gly Glu Asn Pro Ala Ala Thr Met Thr Glu Gin 10 15 20

GGT GAA GAT ATC ACT ACG AAG AAA GAC AGA GGA GTA TTA AAG ATT GTC 271 Gly Glu Asp lie Thr Thr Lys Lys Asp Arg Gly Val Leu Lys lie Val 25 30 35

AAA AGA GTG GGG ACT AGT GAC GAG GCC CCA ATG TTT GGT GAC AAA GTT 319 Lys Arg Val Gly Thr Ser Asp Glu Ala Pro Met Phe Gly Asp Lys Val 40 45 50

TAT GTC CAC TAC AAA GGG ATG TTG TCA GAT GGA AAG AAG TTT GAT TCC 367 Tyr Val His Tyr Lys Gly Met Leu Ser Asp Gly Lys Lys Phe Asp Ser 55 60 65

AGT CAT GAC AGA AAG AAG CCA TTT GCC TTT AGC CTT GGC CAA GGC CAG 415 Ser His Asp Arg Lys Lys Pro Phe Ala Phe Ser Leu Gly Gin Gly Gin 70 75 80 85

GTT ATC AAA GCC TGG GAC ATT GGG GTG TCT ACT ATG AAG AAA GGC GAG 463 Val lie Lys Ala Trp Asp lie Gly Val Ser Thr Met Lys Lys Gly Glu 90 95 100

ATC TGC CAT TTA TTA TGT AAA CCA GAA TAT GCT TAT GGC TCG GCT GGC 511 lie Cys His Leu Leu Cys Lys Pro Glu Tyr Ala Tyr Gly Ser Ala Gly 105 110 115

CAC CTC CAA AAA ATT CCA TCA AAT GCA ACT CTC TTT TTT GAG ATT GAG 559 His Leu Gin Lys lie Pro Ser Asn Ala Thr Leu Phe Phe Glu He Glu 120 125 130

CTC CTT GAT TTC AAA GGT GAG GAT TTA TTT GAA GAT TCA GGC GTT ATC 607 Leu Leu Asp Phe Lys Gly Glu Asp Leu Phe Glu Asp Ser Gly Val He 135 140 145

CGT AGA ATC AAA CGG AAA GGC GAG GGA TAC TCA AAC CCA AAC GAA GGA 655 Arg Arg He Lys Arg Lys Gly Glu Gly Tyr Ser Asn Pro Asn Glu Gly 150 155 160 165

GCA ACG GTA AAA GTC CAC CTG GAA GGC TGC TGT GGT GGA AGG ACA TTT 703 Ala Thr Val Lys Val His Leu Glu Gly Cys Cys Gly Gly Arg Thr Phe 170 175 180

GAT TGC CGA GAT GTG GTG TTC GTT GTT GGG GAA GGA GAA GAC CAC GAC 751 Asp Cys Arg Asp Val Val Phe Val Val Gly Glu Gly Glu Asp His Asp 185 190 195

ATT CCG ATT GGG ATC GAC AAA GCC CTG GTG AAG ATG CAG AGA GAA GAA He Pro He Gly He Asp Lys Ala Leu Val Lys Met Gin Arg Glu Glu 200 205 210

CAG TGT ATT CTA TAT CTT GGA CCA CGC TAT GGT TTT GGA GAA GCC GGG Gin Cys He Leu Tyr Leu Gly Pro Arg Tyr Gly Phe Gly Glu Ala Gly 215 220 225

AAG CCT AAG TTT GGC ATT GAC CCC AAT GCT GAG CTT ATG TAC GAG GTC 895 Lys Pro Lys Phe Gly He Asp Pro Asn Ala Glu Leu Met Tyr Glu Val 230 235 240 245

ACC CTT AAG AGC TTC GAG AAG GCC AAA GAA TCT TGG GAG ATG GAC ACC 943 Thr Leu Lys Ser Phe Glu Lys Ala Lys Glu Ser Trp Glu Met Asp Thr 250 255 260

AAA GAA AAG CTG ACG CAG GCT GCC ATC GTG AAA GAG AAG GGA ACT GTG 991 Lys Glu Lys Leu Thr Gin Ala Ala He Val Lys Glu Lys Gly Thr Val 265 270 275

TAC TTC AAG GGA GGC AAG TAC ACG CAG GCC GTG ATT CAG TAC AGG AAG 1039 Tyr Phe Lys Gly Gly Lys Tyr Thr Gin Ala Val He Gin Tyr Arg Lys 280 285 290

ATA GTG TCC TGG CTG GAG ATG GAA TAC GGC CTG TCA GAG AAG GAG TCC 1087 He Val Ser Trp Leu Glu Met Glu Tyr Gly Leu Ser Glu Lys Glu Ser 295 300 305

AAA GCC TCA GAG TCG TTC CTC CTC GCA GCC TTC CTG AAC CTG GCC ATG 1135 Lys Ala Ser Glu Ser Phe Leu Leu Ala Ala Phe Leu Asn Leu Ala Met 310 315 320 325

TGC TAC CTG AAG CTC CGA GAG TAC AAC AAA GCC GTG GAG TGC TGC GAC 1183 Cys Tyr Leu Lys Leu Arg Glu Tyr Asn Lys Ala Val Glu Cys Cys Asp 330 335 340

AAG GCC CTT GGA CTG GAC AGT GCC AAT GAG AAA GGC TTG TAC AGA AGG 1231 Lys Ala Leu Gly Leu Asp Ser Ala Asn Glu Lys Gly Leu Tyr Arg Arg 345 350 355

GGC GAG GCC CAG CTG CTC ATG AAT GAC TTT GAG TCG GCC AAG GGC GAC 1279 Gly Glu Ala Gin Leu Leu Met Asn Asp Phe Glu Ser Ala Lys Gly Asp 360 365 370

TTC GAG AAG GTG TTG GCA GTC AAT CCT CAG AAC AGG GCC GCT CGC CTG 1327 Phe Glu Lys Val Leu Ala Val Asn Pro Gin Asn Arg Ala Ala Arg Leu 375 380 385

CAG ATC TCC ATG TGC CAG AGG AAG GCG AAG GAG CAC AAC GAG CGG GAC 1375 Gin He Ser Met Cys Gin Arg Lys Ala Lys Glu His Asn Glu Arg Asp 390 395 400 405

CGC AGG GTG TAC GCC AAC ATG TTC AAG AAG TTC GCA GAG CGG GAC GCA 1423 Arg Arg Val Tyr Ala Asn Met Phe Lys Lys Phe Ala Glu Arg Asp Ala 410 415 420

AAG GAG GAA GCC AGC AAA GCT GGG AGC AAG AAG GCT GTA GAA GGA GCC 1471 Lys Glu Glu Ala Ser Lys Ala Gly Ser Lys Lys Ala Val Glu Gly Ala 425 430 435

GCT GGC AAA CAA CAC GAG AGT CAG GCC ATG GAA GAA GGA AAG GCC AAA 1519 Ala Gly Lys Gin His Glu Ser Gin Ala Met Glu Glu Gly Lys Ala Lys 440 445 450 GGC CAT GTA TGACGCTGCG CCACGGAGGG AAGAGAGTCC TAATGAACTC 1

Gly His Val 455

GGCCCTCCTC GCTGGGCTCG CCTCCAACTC AGGACTGAAC AGTGTTTAGT GTAAGGTTTG 1

TTACAGTCTC TGTGATTCTG GAAGCAAATG GCATACCAGT AGCTTCCCAA ATGACCACCT 1

GCTGCTGCGG GGGGGTGGGG GTGGGGGACA TGCCAGGAAA CAGCAGAGAA GGCCGCTGGT 1

GTGAAGAGAC CAGGCCAGCA GCTCAGTCCA GCCCATTTCA GTTTGTCACC TTTCAGTGTC 1

CAGCACAGCA TCCCTGTGAA CCTAGGGCCC AGCTGCTGTG GGTTCTACAT CGGCACTAGG 1

GTCACACTGC AGAAACCGTT GATAAAACAA ACTCAGTGAT CTCTGCTTTC CTATTGGTGG 1

GCATGGCAGG GGCGGGTGAT GAGATTTGCT TAGCACTGAC TGACTGGCCT GCTAAGAACA 1

CAAGCCCACA GCCAGGGGCT CCCTGGTCCA CAGCTGGGTC TCAGGCCCCT TACCTGCCTT 2

CCAAGTCCTT TCGCAGACTC TTGAGTGTGG CTTTCTGTCC TAGCCAGCAT GTCCCACAGA 2

CTCTGTTGTT CCTCCAACGC CCGTCATTAG TGACAGCTTT CTCTCTGAGT TTCTGTGGTG 2

TGGAGAGTGG GTAGAAGTAG GTTTATCTTT CCCGCTGTCT GCCCCACTCA AGGACGAT 2

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 456 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Met Thr Thr Asp Glu Gly Thr Ser Asn Asn Gly Glu Asn Pro Ala Ala 1 5 10 15

Thr Met Thr Glu Gin Gly Glu Asp He Thr Thr Lys Lys Asp Arg Gly 20 25 30

Val Leu Lys He Val Lys Arg Val Gly Thr Ser Asp Glu Ala Pro Met 35 40 45

Phe Gly Asp Lys Val Tyr Val His Tyr Lys Gly Met Leu Ser Asp Gly 50 55 60

Lys Lys Phe Asp Ser Ser His Asp Arg Lys Lys Pro Phe Ala Phe Ser 65 70 75 80

Leu Gly Gin Gly Gin Val He Lys Ala Trp Asp He Gly Val Ser Thr 85 90 95 Met Lys Lys Gly Glu He Cys His Leu Leu Cys Lys Pro Glu Tyr Ala 100 105 110

Tyr Gly Ser Ala Gly His Leu Gin Lys He Pro Ser Asn Ala Thr Leu 115 120 125

Phe Phe Glu He Glu Leu Leu Asp Phe Lys Gly Glu Asp Leu Phe Glu 130 135 140

Asp Ser Gly Val He Arg Arg He Lys Arg Lys Gly Glu Gly Tyr Ser 145 150 155 160

Asn Pro Asn Glu Gly Ala Thr Val Lys Val His Leu Glu Gly Cys Cys 165 170 175

Gly Gly Arg Thr Phe Asp Cys Arg Asp Val Val Phe Val Val Gly Glu 180 185 190

Gly Glu Asp His Asp He Pro He Gly He Asp Lys Ala Leu Val Lys 195 200 205

Met Gin Arg Glu Glu Gin Cys He Leu Tyr Leu Gly Pro Arg Tyr Gly 210 215 220

Phe Gly Glu Ala Gly Lys Pro Lys Phe Gly He Asp Pro Asn Ala Glu 225 230 235 240

Leu Met Tyr Glu Val Thr Leu Lys Ser Phe Glu Lys Ala Lys Glu Ser 245 250 255

Trp Glu Met Asp Thr Lys Glu Lys Leu Thr Gin Ala Ala He Val Lys 260 265 270

Glu Lys Gly Thr Val Tyr Phe Lys Gly Gly Lys Tyr Thr Gin Ala Val 275 280 285

He Gin Tyr Arg Lys He Val Ser Trp Leu Glu Met Glu Tyr Gly Leu 290 295 300

Ser Glu Lys Glu Ser Lys Ala Ser Glu Ser Phe Leu Leu Ala Ala Phe 305 310 315 320

Leu Asn Leu Ala Met Cys Tyr Leu Lys Leu Arg Glu Tyr Asn Lys Ala 325 330 335

Val Glu Cys Cys Asp Lys Ala Leu Gly Leu Asp Ser Ala Asn Glu Lys 340 345 350

Gly Leu Tyr Arg Arg Gly Glu Ala Gin Leu Leu Met Asn Asp Phe Glu 355 360 365

Ser Ala Lys Gly Asp Phe Glu Lys Val Leu Ala Val Asn Pro Gin Asn 370 375 380

Arg Ala Ala Arg Leu Gin He Ser Met Cys Gin Arg Lys Ala Lys Glu 385 390 395 400 His Asn Glu Arg Asp Arg Arg Val Tyr Ala Asn Met Phe Lys Lys Phe 405 410 415

Ala Glu Arg Asp Ala Lys Glu Glu Ala Ser Lys Ala Gly Ser Lys Lys 420 425 430

Ala Val Glu Gly Ala Ala Gly Lys Gin His Glu Ser Gin Ala Met Glu 435 440 445

Glu Gly Lys Ala Lys Gly His Val 450 455

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1619 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: unknown

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(vii) IMMEDIATE SOURCE:

(B) CLONE: hm51-35

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 154..1524

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

GGGCCGGCTC GCGGGCGCTG CCAGTCTCGG GCGGCGGTGT CCGGCGCGCG GGCGGCCTGC

TGGGCGGGCT GAAGGGTTAG CGGAGCACGG GCAAGGCGGA GAGTGACGGA GTCGGCGAGC

CCCCGCGGCG ACAGGTTCTC TACTTAAAAG ACA ATG ACT ACT GAT GAA GGT GCC

Met Thr Thr Asp Glu Gly Ala 1 5

AAG AAC AAT GAA GAA AGC CCC ACA GCC ACT GTT GCT GAG CAG GGA GAG Lys Asn Asn Glu Glu Ser Pro Thr Ala Thr Val Ala Glu Gin Gly Glu 10 15 20

GAT ATT ACC TCC AAA AAA GAC AGG GGA GTA TTA AAG ATT GTC AAA AGA Asp He Thr Ser Lys Lys Asp Arg Gly Val Leu Lys He Val Lys Arg 25 30 35

GTG GGG AAT GGT GAG GAA ACG CCG ATG ATT GGA GAC AAA GTT TAT GTC Val Gly Asn Gly Glu Glu Thr Pro Met He Gly Asp Lys Val Tyr Val 40 45 50 55

CAT TAC AAA GGA AAA TTG TCA AAT GGA AAG AAG TTT GAT TCC AGT CAT His Tyr Lys Gly Lys Leu Ser Asn Gly Lys Lys Phe Asp Ser Ser His

60 65 70

GAT AGA AAT GAA CCA TTT GTC TTT AGT CTT GGC AAA GGC CAA GTC ATC 414

Asp Arg Asn Glu Pro Phe Val Phe Ser Leu Gly Lys Gly Gin Val He

75 80 85

AAG GCA TGG GAC ATT GGG GTG GCT ACC ATG AAG AAA GGA GAG ATA TGC 462

Lys Ala Trp Asp He Gly Val Ala Thr Met Lys Lys Gly Glu He Cys

90 95 100

CAT TTA CTG TGC AAA CCA GAA TAT GCA TAT GGC TCG GCT GGC AGT CTC 510

His Leu Leu Cys Lys Pro Glu Tyr Ala Tyr Gly Ser Ala Gly Ser Leu

105 110 115

CCT AAA ATT CCC TCG AAT GCA ACT CTC TTT TTT GAG ATT GAG CTC CTT 558

Pro Lys He Pro Ser Asn Ala Thr Leu Phe Phe Glu He Glu Leu Leu

120 125 130 135

GAT TTC AAA GGA GAG GAT TTA TTT GAA GAT GGA GGC ATT ATC CGG AGA 606

Asp Phe Lys Gly Glu Asp Leu Phe Glu Asp Gly Gly He He Arg Arg

140 145 150

ACC AAA CGG AAA GGA GAG GGA TAT TCA AAT CCA AAC GAA GGA GCA ACA 654

Thr Lys Arg Lys Gly Glu Gly Tyr Ser Asn Pro Asn Glu Gly Ala Thr

155 160 165

GTA GAA ATC CAC CTG GAA GGC CGC TGT GGT GGA AGG ATG TTT GAC TGC 702

Val Glu He His Leu Glu Gly Arg Cys Gly Gly Arg Met Phe Asp Cys

170 175 180

AGA GAT GTG GCA TTC ACT GTG GGC GAA GGA GAA GAC CAC GAC ATT CCA 750

Arg Asp Val Ala Phe Thr Val Gly Glu Gly Glu Asp His Asp He Pro

185 190 195

ATT GGA ATT GAC AAA GCT CTG GAG AAA ATG CAG CGG GAA GAA CAA TGT 798

He Gly He Asp Lys Ala Leu Glu Lys Met Gin Arg Glu Glu Gin Cys

200 205 210 215

ATT TTA TAT CTT GGA CCA AGA TAT GGT TTT GGA GAG GCA GGG AAG CCT 846

He Leu Tyr Leu Gly Pro Arg Tyr Gly Phe Gly Glu Ala Gly Lys Pro

220 225 230 AAA TTT GGC ATT GAA CCT AAT GCT GAG CTT ATA TAT GAA GTT ACA CTT Lys Phe Gly He Glu Pro Asn Ala Glu Leu He Tyr Glu Val Thr Leu 235 240 245

AAG AGC TTC GAA AAG GCC AAA GAA TCC TGG GAG ATG GAT ACC AAA GAA Lys Ser Phe Glu Lys Ala Lys Glu Ser Trp Glu Met Asp Thr Lys Glu 250 255 260

AAA TTG GAG CAG GCT GCC ATT GTC AAA GAG AAG GGA ACC GTA TAC TTC Lys Leu Glu Gin Ala Ala He Val Lys Glu Lys Gly Thr Val Tyr Phe 265 270 275

AAG GGA GGC AAA TAC ATG CAG GCG GTG ATT CAG TAT GGG AAG ATA GTG Lys Gly Gly Lys Tyr Met Gin Ala Val He Gin Tyr Gly Lys He Val 280 285 290 295

TCC TGG TTA GAG ATG GAA TAT GGT TTA TCA GAA AAG GAA TCG AAA GCT Ser Trp Leu Glu Met Glu Tyr Gly Leu Ser Glu Lys Glu Ser Lys Ala 300 305 310

TCT GAA TCA TTT CTC CTT GCT GCC TTT CTG AAC CTG GCC ATG TGC TAC Ser Glu Ser Phe Leu Leu Ala Ala Phe Leu Asn Leu Ala Met Cys Tyr 315 320 325

CTG AAG CTT AGA GAA TAC ACC AAA GCT GTT GAA TGC TGT GAC AAG GCC Leu Lys Leu Arg Glu Tyr Thr Lys Ala Val Glu Cys Cys Asp Lys Ala 330 335 340

CTT GGA CTG GAC AGT GCC AAT GAG AAA GGC TTG TAT AGG AGG GGT GAA Leu Gly Leu Asp Ser Ala Asn Glu Lys Gly Leu Tyr Arg Arg Gly Glu 345 350 355

GCC CAG CTG CTC ATG AAC GAG TTT GAG TCA GCC AAG GGT GAC TTT GAG Ala Gin Leu Leu Met Asn Glu Phe Glu Ser Ala Lys Gly Asp Phe Glu 360 365 370 375

AAA GTG CTG GAA GTA AAC CCC CAG AAT AAG GCT GCA AGA CTG CAG ATC Lys Val Leu Glu Val Asn Pro Gin Asn Lys Ala Ala Arg Leu Gin He 380 385 390

TCC ATG TGC CAG AAA AAG GCC AAG GAG CAC AAC GAG CGG GAC CGC AGG Ser Met Cys Gin Lys Lys Ala Lys Glu His Asn Glu Arg Asp Arg Arg 395 400 405

ATA TAC GCC AAC ATG TTC AAG AAG TTT GCA GAG CAG GAT GCC AAG GAA He Tyr Ala Asn Met Phe Lys Lys Phe Ala Glu Gin Asp Ala Lys Glu 410 415 420

GAG GCC AAT AAA GCA ATG GGC AAG AAG ACT TCA GAA GGG GTC ACT AAT Glu Ala Asn Lys Ala Met Gly Lys Lys Thr Ser Glu Gly Val Thr Asn 425 430 435

GAA AAA GGA ACA GAC AGT CAA GCA ATG GAA GAA GAG AAA CCT GAG GGC Glu Lys Gly Thr Asp Ser Gin Ala Met Glu Glu Glu Lys Pro Glu Gly 440 445 450 455 CAC GTA TGACGCCACG CCAAGGAGGG AAGAGTCCCA GTGAACTCGG CCCCTCCTCA 1574 His Val

ATGGGCTTTC CCCCAACTCA GGACAGAACA GTGTTTAATG TAAAG 1619

(2) INFORMATION FOR SEQ ID N0:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 457 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:

Met Thr Thr Asp Glu Gly Ala Lys Asn Asn Glu Glu Ser Pro Thr Ala 1 5 10 15

Thr Val Ala Glu Gin Gly Glu Asp He Thr Ser Lys Lys Asp Arg Gly 20 25 30

Val Leu Lys He Val Lys Arg Val Gly Asn Gly Glu Glu Thr Pro Met 35 40 45

He Gly Asp Lys Val Tyr Val His Tyr Lys Gly Lys Leu Ser Asn Gly 50 55 60

Lys Lys Phe Asp Ser Ser His Asp Arg Asn Glu Pro Phe Val Phe Ser 65 70 75 80

Leu Gly Lys Gly Gin Val He Lys Ala Trp Asp He Gly Val Ala Thr 85 90 95

Met Lys Lys Gly Glu He Cys His Leu Leu Cys Lys Pro Glu Tyr Ala 100 105 110

Tyr Gly Ser Ala Gly Ser Leu Pro Lys He Pro Ser Asn Ala Thr Leu 115 120 125

Phe Phe Glu He Glu Leu Leu Asp Phe Lys Gly Glu Asp Leu Phe Glu 130 135 140

Asp Gly Gly He He Arg Arg Thr Lys Arg Lys Gly Glu Gly Tyr Ser 145 150 155 160

Asn Pro Asn Glu Gly Ala Thr Val Glu He His Leu Glu Gly Arg Cys 165 170 175

Gly Gly Arg Met Phe Asp Cys Arg Asp Val Ala Phe Thr Val Gly Glu 180 185 190

Gly Glu Asp His Asp He Pro He Gly He Asp Lys Ala Leu Glu Lys 195 200 205 Met Gin Arg Glu Glu Gin Cys He Leu Tyr Leu Gly Pro Arg Tyr Gly 210 215 220

Phe Gly Glu Ala Gly Lys Pro Lys Phe Gly He Glu Pro Asn Ala Glu 225 230 235 240

Leu He Tyr Glu Val Thr Leu Lys Ser Phe Glu Lys Ala Lys Glu Ser 245 250 255

Trp Glu Met Asp Thr Lys Glu Lys Leu Glu Gin Ala Ala He Val Lys 260 265 270

Glu Lys Gly Thr Val Tyr Phe Lys Gly Gly Lys Tyr Met Gin Ala Val 275 280 285

He Gin Tyr Gly Lys He Val Ser Trp Leu Glu Met Glu Tyr Gly Leu 290 295 300

Ser Glu Lys Glu Ser Lys Ala Ser Glu Ser Phe Leu Leu Ala Ala Phe 305 310 315 320

Leu Asn Leu Ala Met Cys Tyr Leu Lys Leu Arg Glu Tyr Thr Lys Ala 325 330 335

Val Glu Cys Cys Asp Lys Ala Leu Gly Leu Asp Ser Ala Asn Glu Lys 340 345 350

Gly Leu Tyr Arg Arg Gly Glu Ala Gin Leu Leu Met Asn Glu Phe Glu 355 360 365

Ser Ala Lys Gly Asp Phe Glu Lys Val Leu Glu Val Asn Pro Gin Asn 370 375 380

Lys Ala Ala Arg Leu Gin He Ser Met Cys Gin Lys Lys Ala Lys Glu 385 390 395 400

His Asn Glu Arg Asp Arg Arg He Tyr Ala Asn Met Phe Lys Lys Phe 405 410 415

Ala Glu Gin Asp Ala Lys Glu Glu Ala Asn Lys Ala Met Gly Lys Lys 420 425 430

Thr Ser Glu Gly Val Thr Asn Glu Lys Gly Thr Asp Ser Gin Ala Met 435 440 445

Glu Glu Glu Lys Pro Glu Gly His Val 450 455 SEQ ID NO : 5 :

N-succinyl-Ala-X-Pro-Phe-p-nitroanilide

SEQ ID NO:6:

N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide

SEQ ID NO:7:

N-succinyl-Ala-Leu-Pro-Phe-p-nitroanilide

SEQ ID NO:8:

CGGGATCCGG ACAATGACTA CTGATGAG

SEQ ID NO:9:

CGGAATTCCG TCATACATGG CCTTTGGC

SEQ ID NO:10:

YGESQAMEEGKAKGHV

Claims

What is claimed is:

1. An isolated mammalian immunophilin protein characterized as being predominantly expressed in T-lymphocytes and capable of inhibiting calcineurin when complexed to ligand, and functional fragments of said protein.

2. An immunophilin protein according to Claim 1, further characterized as corresponding to mRNA induced by glucocorticoid.

3. An immunophilin protein according to Claim 2 wherein said glucocorticoid is dexamethasone.

4. An immunophilin protein according to Claim 1 wherein the amino acid sequence of said protein is substantially the same as the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:4.

5. An immunophilin protein according to Claim 1 wherein the amino acid sequence of said protein is substantially the same as the amino acid sequence set forth in SEQ ID NO:4.

6. An immunophilin protein according to Claim 1 having the same amino acid sequence as set forth in SEQ ID NO:2 or SEQ ID NO:4.

7. An immunophilin protein according to Claim 1 having the same amino acid sequence as set forth in SEQ ID NO:4.

8. An isolated nucleic acid encoding a protein according to Claim 1.

9. An isolated nucleic acid according to Claim 8 having a contiguous nucleotide sequence substantially the same as nucleotides 161 to 1528 of SEQ ID NO:l, nucleotides 154 to 1524 of SEQ ID NO:3, or degenerate variations thereof.

10. An isolated nucleic acid according to Claim 8 having a contiguous nucleotide sequence substantially the same as nucleotides 161 to 1528 of SEQ ID NO:3.

11. An isolated nucleic acid according to Claim 8 having a contiguous nucleotide sequence substantially the same as set forth in SEQ ID NO:l or SEQ ID NO:3.

12. An isolated nucleic acid according to Claim 8 having a contiguous nucleotide sequence substantially the same as set forth in SEQ ID NO:3.

13. Isolated nucleic acid, or functional fragments thereof encoding the protein of Claim 1, said nucleic acid selected from:

(a) nucleic acid encoding the amino acid sequence set forth in SEQ ID N0:1 or SEQ ID

NO:3;

(b) nucleic acid that hybridizes to the nucleic acid of (a) wherein said nucleic acid encodes biologically active immunophilin protein; or

(c) nucleic acid degenerate with respect to either (a) or (b) above, wherein said nucleic acid encodes biologically active immunophilin protein.

14. A recombinant expression vector comprising nucleic acid according to Claim 8.

15. A host cell containing the vector of Clai 14.

16. An antibody generated against the protein of

Claim 1.

17. An antibody according to Claim 16 wherei said antibody is monoclonal antibody.

18. A method for identifying compounds whic bind to immunophilin protein according to Claim l, sai method comprising:

independently contacting said protein wit labeled immunosuppressive compound, known to bind sai protein, in the presence and absence of test compound t produce reaction mixtures containing protein bound compoun and unbound compound;

separating protein bound compound an unbound compound in each reaction mixture; and

determining the amount of label associate with said protein in the presence of test compound relativ to label associated with said protein in the absence o test compound.

19. A method according to Claim 18 wherein sai separating comprises:

(a) treating protein bound compound an unbound compound with an inert matri material capable of binding unboun compound; and thereafter;

(b) separating protein bound compound fro matrix bound compound.

20. A method for identifying compounds capable of inhibiting the phosphatase activity of calcineurin when said compounds are bound to immunophilin protein of Claim 1, said method comprising:

contacting labeled phosphorylated substrate with protein and with sufficient amounts of calcineurin and calmodulin to produce free labeled phosphate;

repeating the above contacting step in the further presence of test compound; and

determining the amount of free labeled phosphate generated in the presence of protein alone relative to the amount generated in the further presence of test compound.

21. A method for identifying compounds capable of inhibiting phosphatase activity of calcineurin when said compounds are bound to protein of Claim 1, said method comprising:

independently contacting labeled phosphorylated substrate with test compound, increasing amounts of said protein and sufficient amounts of calcineurin and calmodulin to produce reaction mixtures containing free labeled phosphate;

determining the amount of free labeled phosphate produced in each reaction mixture; and

selecting those compounds which cause decreased production of free labeled phosphate in the presence of increasing amounts of said protein.

22. A method according to Claim 21 further comprising treating the reaction mixtures to separate free labeled phosphate from labeled phosphorylated substrate prior to determining the amount of free labeled phosphate.