GB2279349A - Human FKBP-38 protein - Google Patents

Abstract

The gene encoding the human 38kDa FK-506 binding protein is described, as well as the amino acid sequence of the derived protein product. This binding protein is useful, inter alia, for screening FK-506 type compounds useful as immunosuppressive drugs that block allograft rejection by preventing T-cell activation.

Description

TITTLE OF THE INVENTION GENE ENCODING THE HUMAN 38kDa FK-506 BINDING PROTEIN BACKGROUND OF THE INVENTION 1. Field of the Invention The field of the present invention is immunosuppressive drugs that block allograft rejection by preventing T-cell activation.

FK-506, (see USP 4,894,366 for description and synthesis) a neutral macrolide metabolite of S. tsukabaensis, is an important new immunosuppressive drug with 10 to 100 times the potency of the cyclic peptide, cyclosporin A (CsA), (see USP 4,117,118 for description and synthesis) as measured by its efficacy in preventing allograft rejection.

Both FK-506 and CsA exert their inhibitory effects during the signal transduction events leading to T-lymphocyte activation by selectively blocking transcription of the same set of early phase lymphokine genes, including those encoding IL-2, IL-3, IL-4, IFN-y, TNF-a, GM-CSF and c-myc. Although their effects upon T-cell activation are indistinguishable, FK-506 and CsA bind to distinct, abundant (0.1%0.4% of total cellular protein), cytosolic receptors termed the FK-506 binding protein (FKBP-12; Mr=11,400) and cyclophilin (Mr=17,000), respectively.

FKBP-12 is not a lymphoid-specific protein, but is widely distributed in tissues and throughout the phyla. The FKBP-12 protein is a member of a new class of enzymes, collectively called "FKBPs" and termed peptidyl-prolyl isomerases (PPIases) which catalyze isomerization between the cis and trans forms of the Xaa-Pro bond in peptides and proteins. Known FKBPs include FKBP-12, (see USP 5,109,112), FKBP 13 (see PNAS, Vol. 88, p. 6677-6681 1991), FKBP 25 (see Biochem. and Biophys. Res. Comm. Vol. 185 p. 298-303 1992 by G. Wiedderecht et al.), and FKBP 52, which is described in J. Biol.

Chem. 267, pp. 21753-21760 1992 by G. Wiedderecht et al. Rotation about the peptidyl-prolyl bond is one of the predominant slow steps involved in refolding denatured proteins in vitro. Their ubiquity and abundance suggest that FKBPs have an important role in cellular physiology, perhaps in accelerating interconversion between rotational conformers of denatured or recently synthesized proteins. The PPIase activities of all known FKBPs are specifically inhibited by FK-506.

2. Brief Description of the Prior Art FKBP-12 has been purified to homogeneity from human Tlymphocytes from calf thymus. Siekierka et al., Nature, 341, 755-757 (1989); and Harding et al., Nature,341, 758-760 (1989) and cloned by a number of groups. The amino-terminal sequence of human FKBP 12, as deduced from the nucleotide sequence, matches the reported protein sequence except for the addition of an initiator methionine in the clone.

Although it is likely that FKBP 12 is mediating this immunosuppressive activity of FK506, it has not been formally proven by gene disruptions.

SUMMARY OF THE INVENTION The present invention relates to the nucleotide sequence of the cDNA encoding the new human 38kDa FK-506 binding protein homolog (FKBP-38), and to the amino acid sequence of said binding protein derived from said nucleotide sequence. In addition to providing these, it is an object of the present invention to provide a vector containing the DNA sequence encoding FKBP-38, to provide a host cell transformed with said vector containing said DNA sequence, and to provide a recombinant process for making FKBP-38 by culturing said transformed host cell under conditions suitable for the expression of FKBP-38, and recovering FKBP-38.

The present invention also relates to a method of testing chemical compounds in order to identify those which have immunosuppressive activity capable of blocking allograft rejection by preventing T-cell activation, comprising bringing said test chemical compound into contact with FKBP-38 and determining whether said test compound will bind thereto.

It is a further object of the present invention to provide purified nucleic acid probes which will enable the determination of FKBP-38 expression in various species and in various tissues and cells within the selected species.

By this invention there is provided a DNA sequence coding for the human 38kDa FK-506 binding protein.

Specifically provided is DNA sequence as shown below for coding for the human 38kDa FK-506 binding protein: (SEQ. ID No. 1) GATTCCCCTC ACCCTCTGCA TCCTCAACCC CATCCAGTAC CTCGAAGTCC TCGAGCGGTG 60 GGACCCCGGC GGGTGAGGAG GAAGAGGAGG AGGAAGAGGA GGAAGAGGAT GACCTGAGTG 120 AGCTGCCACC GCTGGAGGAC ATGGGACAAC CCCCGGCGGA GGAGGCTGAG CAGCCTGSGG 180 CCCTSGCCCG AGAGTTCCTT GCTGCCATGG AGCCCGAGCC CGCCCCAGCC CCGGCCCCAG 240 AAGAGTGGCT GGACATTCTG GGGAACGSGC TGTTGAGGAA GAAGACGCTG GTCCCAGGGC 300 CGCCAGGTTC GAGCCGCCCG GTCAAGGGCC AGGTGGTCAC CGTACATCTG CAGACGTCGC 360 TGGAGAATGG CACACGGGTG CAGGAGGAGC CGGAGCTGGT GTTCACTCTG GGTGACTGTG 420 ACGTCATCCA GGCCCTGGAT CTCAGTGTCC CACTCATGGA CGTGGGGGAG ACGGCCATGG 480 TCACTGCTGA CTCCAAGTAC TGCTACGGCC CCCAAGGCAG GAGCCCATAC ATCCCCCCGC 540 ACGCGGCCCT GTGCCTGGAG GTGACCCTGA AGACGGCTGT GGACGGGCCT GACCTGGAGA 600 TGCTCACGGG GCAGGAGCGC GTGGCCCTGG CCAACCGGAA GCGGGAGTGC GGCAACGCCC 660 ACTACCAGCG GGCGGACTTC GTCCTGGCCG CCAACTCCTA CGACCTCGCC ATCAAGGCTA 720 TCACCTCCAG CGCCAAAGTG GACATGACGT TCGAGGAGGA GGCACAGCTC CTGCAGTTGA 780 AGGTGAAGTG TCTGAACAAC CTGGCGGCCT CGCAGCTGAA GCTCGACCAC TACCGCGCAG 840 CCCTGCGCTC CTGCAGCCTT GTGCTGGAGC ACCAGCCAGA CAACATCAAG GCTCTCTTCC 900 GCAAGGGCAA GGTGCTGGCC CAGCAGSGSw AGTACAGTGA GGCCATCCCC ATCCTGAGGG 960 CAGCCCTGAA GCTGGAACCT TCCAACAAGA CGATCCACGC AGAGCTCTCA AAGCTGGTGA 1020 AGAAGCATGC GGCGCAGCGG AGCACGGAGA CCGCCTTGTA CCGGAAAATG CTGGGCAACC 1080 CCAGCCGGCT GCCTGCTAAG TGCCCTGGCA AGSGTGCCTG GTCCATCCCA TGGAAGTGGC 1140 TGTTTGGGGC GACTGCTGTT GCCTTGGGGG GTGTGGCACT CTCTGTGGTC ATCGCTGCCA 1200 GGAACTGACC ACCTAGGTGG CTGCCACCCC CTCTGCACAC CATGGACCCT GCCCTGCGCT 1260 CCCCAACTCC CCCAGGCTCC CTGTCCACTG CCCTCCCTGG TCTGGCCCCC TCCTCCGGGT 1320 TAGGGGAGCA AGGATTGGGG GTCGTGCAGC CCAGCCAGCA GGAGGGACTG AGGCCCTCTA 1380 GGAGGAAAGC CCAGAGGGAG GGGGCCCTCA TTCCTTCAGA CCCAGTTTTC CCCCACCCTC 1440 CTTACCCCGC TGGGCTAGGT CTCCGCCAGG GCTGGCCTCA GTTTCTCCTC AACAGGCCTG 1500 GGGGCAGCCC TTCCCCTGCC TAGTCCCCGC CTGAGTGCCA GCCCCCCACC CCGCCTGCCG 1560 CCCCCTGTCC AGGTTCCCTC CCCGCCACAG TGAAATAAAG CATCCCACCC TGCAAAAAAA 1620 AAAAAAAAAA AAAAGGAATT C , and variants thereof. 1641 Also provided is the human 38kDa FK-506 binding protein having the amino acid sequence as shown below: (SEQ. ID No. 2) Met Gly Gln Pro Pro Ala Glu Glu Ala Glu Gln Pro Gly Ala Leu Ala 5 10 15 Arg Glu Phe Leu Ala Ala Met Glu Pro Glu Pro Ala Pro Ala Pro Ala 20 25 30 Pro Glu Glu Trp Leu Asp Ile Leu Gly Asn Gly Leu Leu Arg Lys Lys 35 40 45 Thr Leu Val Pro Gly Pro Pro Gly Ser Ser Arg Pro Val Lys Gly Gln 50 55 60 Val Val Thr Val His Leu Gln Thr Ser Leu Glu Asn Gly Thr Arg Val 65 70 75 80 Gln Glu Glu Pro Glu Leu Val Phe Thr Leu Gly Asp Cys Asp Val Ile 85 90 95 Gln Ala Leu Asp Leu Ser Val Pro Leu Met Asp Val Gly Glu Thr Ala 100 105 110 Met Val Thr Ala Asp Ser Lys Tyr Cys Tyr Gly Pro Gln Gly Arg Ser 115 120 125 Pro Tyr Ile Pro Pro His Ala Ala Leu Cys Leu Glu Val Thr Leu Lys 130 135 140 Thr Ala Val Asp Gly Pro Asp Leu Glu Met Leu Thr Gly Gln Glu Arg 145 150 155 160 Val Ala Leu Ala Asn Arg Lys Arg Glu Cys Gly Asn Ala His Tyr Gln 165 170 175 Arg Ala Asp Phe Val Leu Ala Ala Asn Ser Tyr Asp Leu Ala Ile Lys - 180 185 190 Ala Ile Thr Ser Ser Ala Lys Val Asp Met Thr Phe Glu Glu Glu Ala 195 200 205 Gln Leu Leu Gln Leu Lys Val Lys Cys Leu Asn Asn Leu Ala Ala Ser 210 c 215 220 Gln Leu Lys Leu Asp His Tyr Arg Ala Ala Leu Arg Ser Cys Ser Leu 225 230 235 240 Val Leu Glu His Gln Pro Asp Asn Ile Lys Ala Leu Phe Arg Lys Gly 245 250 255 Lys Val Leu Ala Gln Gln Gly Glu Tyr Ser Glu Ala Ile Pro Ile Leu 260 265 270 Arg Ala Ala Leu Lys Leu Glu Pro Ser Asn Lys Thr Ile His Ala Glu 275 280 285 Leu Ser Lys Leu Val Lys Lys His Ala Ala Gln Arg Ser Thr Glu Thr 290 295 300 Ala Leu Tyr Arg Lys Met Leu Gly Asn Pro Ser Arg Leu Pro Ala Lys 305 310 315 320 Cys Pro Gly Lys Gly Ala Trp Ser Ile Pro Trp Lys Trp Leu Phe Gly 325 330 335 Ala Thr Ala Val Ala Leu Gly Gly Val Ala Leu Ser Val Val Ile Ala 340 345 350 Ala Arg Asn, and variants thereof.

355 Further provided are a vector containing the above DNA sequence; a microbial host transformed by the same vector containing the DNA sequence coding for the human 38 kDa FK-506 binding protein; a process for the preparation of the human 38 kDa FK-506 binding protein comprising culturing the above transformed host under conditions suitable for the expression of the human 38 kDa FK-506 binding protein, and recovering said binding protein; human 38 kDa FK-506 binding protein made by the process and a method of testing chemical compounds in order to identify those which have immunosuppressive activity capable of blocking allograft rejection by preventing T-cell activation, comprising bringing said test chemical compound into contact with the human 38 kDa FK-506 binding protein and determining whether said test compound will bind thereto.

DESCRWflON OF THE DRAWING Fig. 1 Nucleotide sequence of the cDNA is illustrated encoding the human FK-506 binding protein, FKBP-38. The derived amino acid sequence is shown below the nucleotide sequence.

DETAILED DESCRIPTION OF THE INVENTION The proteins, DNA sequences of the disclosed invention include variations, on the disclosed sequence or sequences which conserve the activity of the disclosed sequence or sequences, including fragments or subunits, naturally occurring mutations, allelic variants, randomly generated artificial mutants, and microheterogeneous forms.

Fragments or subunits refers to any portion of the sequence which contains fewer bases in the DNA sequence, or fewer amino acids than the complete protein, e.g. partial sequences excluding portions at the N- and/or C-termini of the complete protein.

Also included in the term "variants" are Microheterogeneous forms, which refers is to a single gene product, that is a protein produced from a single gene unit of DNA, which is structurally modified following translation. These structural modifications, however, do not result in any significant alterations of the activity of the protein. The modifications may take place either in vivo or during the isolation purification process. In vivo modification may result in, but is not limited to, acetylation at the N-terminus, proteolysis, glycosylation or phosphorylation. Proteolysis may include exoproteolysis, wherein one or more terminal amino acids are sequentially, enzymatically cleaved to produce microheterogeneous forms having fewer amino acids than the original gene product.

Proteolysis may also include endoproteolytic modification that results from the action of endoproteases that cleave the peptide at specific sequence locations. Similar modifications, especially proteolytic modifications, can occur during the purification process which may result in the production of microheterogeneous forms.

Also included are hybrid proteins, such as fusion proteins or proteins resulting from the expression of multiple genes within the expression vector, and may include a polypeptide having the specific activity of a disclosed protein linked by peptide bonds to a second polypeptide.

It will be understood that other variants of proteins of the present invention which conserve activity are included, especially variants that differ from the isolated proteins only by conservative amino acid substitution. Conservative amino acid substitutions are defined as "sets" in Table 1 of Taylor, W.R., J. Mol. Biol., Vol. 188, p 233 (1986).

Recombinant DNA technology may be used to produce proteins of the invention. This technology allows segments of genetic information, DNA, from different cells, and usually from different organisms, to be joined end-to-end outside the organisms from which the DNA was obtained and to incorporate this hybrid DNA into a cell that will allow the production of the protein for which the original DNA encodes. Genetic information, DNA or mRNA, is isolated and incorporated into an appropriate cloning vector, and transduced into an appropriate host cell.

Cloning vectors useful for this technology include a DNA sequence which accommodates specific experimental foreign DNA. The vectors are introduced into host cells that can exist in a stable manner and express the protein dictated by the experimental DNA. Cloning vectors may include plasmids, bacteriophage, viruses and cosmids.

Expression vectors are DNA sequences that are required for the transcription of cloned copies of genes and the translation of their mRNAs in an appropriate host. These vectors can express either procaryotic or eucaryotic genes in a variety of cells such as bacteria, yeast, insect and mammalian cells.

Proteins may also be expressed in a number of virus systems. A suitably constructed expression vector contains an origin of replication for autonomous replication in host cells, selective markers, a limited nuinber of useful restriction enzyme sites, a high copy number, and strong promoters. Promoters are DNA sequences that direct RNA polymerase to bind to DNA and initiate RNA synthesis; strong promoters cause such initiation at high frequency. Expression vectors may include, but are not limited to cloning vectors, modified cloning vectors and specifically designed plasmids or viruses.

Identification of a 38 kDa FKBP Homolog. FKBP38 A reduced-stringency PCR molecular method to identify novel FK-506 binding proteins and homologs was developed in order to complement the biochemical approaches such as affinity chromatography. This approach was analogously based upon a report by Buck & Axel (Cell 65, 175) who identified novel ss-adrenergic odorant receptors using PCR primers specific for conserved regions of known ss-adrenergic receptors.

The following FKBP DNA sequences were aligned by computer: human FKBP25, human FKBP13, human FKBP12, bovine FKBP12, D. melanogaster FKBP12, S. cerevisie FKBP12, N. crassa FKBP12, an N. meningitidis FKBP homolog, and a P. aeroginosa FKBP homolog. Degenerate inosine-containing PCR primers were synthesized in order to cover some of the most conserved regions of these FKBPs.

Six "sense" and six "anti-sense" EcoRI-tailed PCR primers were synthesized. The numbers in parentheses refer to the names for each of the primers.

The first set of two sense primers covered His26 - Gly35 of human FKBP12. They are (when N=inosine): 5'-GGAATTCCTGGTAYACNGGNAHNYTNGANRAYGG-3', (5'4t1) (SEQ ID No. 3) and 5'-GGAATTCCCAYTAYACNGGNAHNYTNGANRAYGG-3', (5'42) (SEQ ID No. 4).

The second set of two sense primers covered Gly29 Asp38 of human FKBP12. They are: 5'-GGAATTCCGGNAHNYTNGANGAYGGNAMNR WITTYGA-3', (5'43) (SEQ ID No. 5) and 5'-GGAATTCCGGNAHNYTNGANGAYGGNCARR WITTYGA-3', (5'44) (SEQ ID No. 6).

The third set of two sense primers covered Gln54 - Trp60 and Gln54 - Asp61 of human FKBP12. They are: 5'-GGAATTCCSRNVARGTNATNMRNGGNTGG-3', (5'45) (SEQ ID No. 7) and 5'-GGAATTCCSRNVARGTNATNMRNGGNTGGGA-3', (5'46) (SEQ ID No. 8).

The first set of two anti-sense PCR primers covered Gly59 - Leu65 or Val56 - Gly63 of human FKBP12. They are: 5'-GGAATTCCNRSNABNSCNWSNTCCCANCC-3', (3'45) (SEQ ID No. 9) and 5'-GGAATTCCNSCNWSNTCCCANCCNYKNATNAC-3', (3'-#6) (SEQ ID No. 10).

The second set of anti-sense PCR primers covered Ile92 Leu98 of human FKBP12. They are: 5'-GGAATTCCARNKTNGMNYBNGSNGGNAT-3', (3'43) (SEQ ID No. 11) and 5'-GGAATTCCARNKTNGMNYBNCCNGGNAT-3', (3'44) (SEQ ID No. 12).

The third set of anti-sense PCR primers covered Leu99 LeulOS. They are: 5'-GGAATTCCNARYTCNRYNTCRAANRYNAG-3', (3'41) (SEQ ID No. 13) and S-GGAATTCCNARYTCNRYNTCRAANRYNAA-3', (3'42) (SEQ ID No. 14).

JURKAT polyA+ RNA was reverse transcribed with AMV reverse transcriptase and the single-stranded cDNA was subjected to amplification with all 36 possible combinations of the above primers in 36 separate PCR reactions. The PCR conditions were: 48 cycles of the following sequences of temperatures: 95" for 30", 55" for 3'30", and 72" for 30". The resulting PCR products were subjected to electrophoresis on a 4% agarose gel and products of the appropriate size were purified. The purified products were digested with EcoR1, purified again to remove ends, subcloned into the EcoRI site of pUC19, and the 36 ligation products separately transformed into E. coli SURE competent cells.Twenty-eight colonies resulting from the ligation of each primer pair was subjected to PCR screening using pUC-specific primers flanking the EcoRI site. The PCR products were digested with EcoRI and analyzed by agarose gel electrophoresis. Depending upon the combination of PCR primers used originally, the products were digested also with EcoRI and Hindi, EcoRI and ScrFI, or EcoRI and TaqI. If fragment sizes resulting from having clones of human FKBP12, human FKBP13, or human FKBP25 were obtained, then those clones were eliminated. Products which had the appropriately sized EcoRI product and which did not give digestion products expected for the other three known human FKBPs would be analyzed further by sequencing.

One transformant that contained a 150 bp PCR product derived from primers 5'#1 and 3'#3 (the 10th transformant analyzed from this primer pair and named 1-3#10) contained an open reading frame (ORF) that plainly encodes a fragment of a previously unidentified FKBP homolog. It is dissimilar at the nucleotide level to any of the known human FKBPs but the translation of the 150 bp fragment (shown below) shows that it is 34%, 27%, and 26% identical at the amino acid level to the corresponding regions within FKBPs 12, 13, & 30, respectively. If conservative amino acid changes are considered, it is greater than 60% similar to human FKB12. The percent identity figures come from the region between the primers and do not include the regions covered by the PCR primers themselves.

A QCGGGTG QGGAGGAGCCGGAGCTGGTGTTCACTCTGGGTGACTGTGAC FThrArgValGI nGI uGI uProGI uLeuVal PheThrLeuGlyAspcysAsp GTCATC Q GGCCCTGGATCTCAGTGTCCCACT QTGGACGTGGGGGAGACG ) Va I I I eGI nAI aLeuAspLeuSer Va I P roLeuMetAspVa I Gl yGI uThr GC CATGGT CTGCTGACTC TGACTCCAAGTACTGC TACGGCC QGCAGG AI Al aMe Val Va I Th r Al aAspSe r Ly sT y rCysT y rGI y P roGI nGI ySe r A rg AGCCCATACATC tSerProTyrl le Transformant 1-3#10 Sequence listings for this transformant are as follows: ACACGGGTGCAGGAGGAGCCGGAGCTGGTGTTCACTCTGGGTGACTGTGAC GTCATCCAGGCCCATCTCAGTCCCACTCAACGAGACG GCCATGGTCACTGCTGACTCCAAGTACTGCTACGGCCCCCAAGGCAGCAGG AGCCCATACATC (SEQ.ID NO. 15) ThrArgValGlnGluGluProGluLeuValPheThrLeuGlyAspCysAsp VallleGlnAlaLeuAspLeuSerValProLeuMetAspValGlyGluThr AlaMetValThrAlaAspSerLysTyrCysTyrGlyProGlnGlySerArg SerProTyrlle (SEQ. ID NO. 16) The insert from the plasmid containing 1-3#10 (the PCR product encoding the potential FKBP homolog) was excised from the plasmid by digestion with EcoRI, gel purified, and labeled with a 32p dATP by random priming.The labeled DNA was used to probe an oligo-dT primed JURKAT cell cDNA library in lambda gtlO. 1.2 million phage were screened under high stringency conditions (50% fomlamidé, SXSSC, 1X Denhardt's, 0.1% SDS, 100 Rg/ml denatured, sheared salmon sperm DNA @ 42C) with the labeled probe. Three phage were found to reproducibly hybridize to the probe through four rounds of re-screening. All three phage were plaque-purified and the inserts excised with EcoR1 from DNA prepared from plate lysates of the positive lambda clones. The gel purified inserts were subcloned into pUC19.Digestion of rapid lysate DNA prepared from the bacterial transformants revealed that one of the phage inserts was about 600 bp in length while the other two inserts were about 1.6 kb in length.

Sequencing revealed that all three of the cDNAs contained the identical sequence found in the PCR product thus indicating that the 600 bp cDNA was most likely incomplete. One of the 1.6 kb clones was chosen for complete sequencing.

The JURKAT cDNA library in lambda gtlO was purchased from Clontech Laboratories, 4030 Fabian Way, Palo Alto, CA 94303.

DNA Sequencing of Phage DNA Inserts Positive phage were plaque-purified and a plug containing a single plaque was placed in 1 ml of SM containing 20 ml of chloroform. After elution of the phage from the plug for 1 hour, the phage were allowed to infect E. coli and plated to near-confluent lysis in LB-top agarose on LB-agarose plates supplemented with lmM MgS04. After bacterial lysis, SM (5 ml) was added to each plate and the plates were shaken for two hours. The SM was transferred to a tube and debris removed by centrifugation. To the supematant, RNAse and DNAse were added to 1 mg/ml, 4 ml of 20% PEG 8000/2M NaCl were added, and the tube incubated on ice for 1.5 hr. Phage were precipitated by centrifugation (10,000 rpm/ 20 min.) in a Beckman JA-20 rotor. The pellet was resuspended in 0.5 ml SM and 5 ml of 10% SDS, 12.5 ml of 0.2 M EDTA (pH 8.0), and 2.6 ml of 10 mg/ml Proteinase K were added. After incubation at 68"C for 1 hr, protein was removed by phenoUchloroform/isoamyl alcohol extraction.

Phage DNA was precipitated upon the addition of 500 ml isopropanol.

After resuspension of the phage DNA, it was digested with EcoRI to remove the insert. The insert was subcloned into pUC19. Sequencing of both DNA strands by the dideoxy method of Biggin et al., Proc. Natl.

Acad. Sci. USA,80, 3963-3965 (1983) was performed directly from denatured plasmid miniprep DNA.

Utility and Application of the FKBP 38 kDa Protein The FKBP kDa 38 protein, per se or immobilized, can be used as a specific binding partner to a variety of binding ligands for diagnostic, purification of investigatory procedures. A preferred immobilizing matrix is cyanogen bromide activated Sepharose (Pharmacia) to which the protein can be covalently linked, forming an affinity chromatography column.

The FKBP 38 protein is of physiological importance because of the high specificity for binding active forms of the immunosuppressant, FK-506. The above described immobilized affinity matrix can be prepared which can reversibly bind FK-506 in a complex, which can be eluted with aqueous buffer reagents of increasing ionic strength. The formed affinity matrix can also be used to detect FK-506like macrolide substances by displacement of tritiated (3H)-FK-506.

This would include FK-520, FK-523, FK-525 and other FK-506 analogs as disclosed in EPO Publication No.0184162.

The matrix also provides a method to identify and/or quantify FK-506 in serum and other body fluids as well as detect FK-506 like cellular constituents which may be natural ligands.

Still further, the matrix is useful in screening candidate chemical structures that, like FK-506, can have immunosuppressive activity and, therefore, is useful in the development of other classes of drugs that function through the action of this protein.

Since the FKDP 38 protein structure is known, oligonucleotide probes can be used to identify the gene thereby allowing the protein to be produced by known recombinant DNA techniques.

Since, the protein has an affinity for the immunosuppressant FK-506, and its active analogs, it or certain derived chemicals and/or natural derivatives thereof, including subfragments of the whole protein, can be used as a specific binding partner for these ligands in numerous receptor binding procedures known in the art.

Similarly, it can be used to purifiy a desired ligand from a composition containing ligand. For example, the protein can be used for purifiying FK-506 or related structures from a yeast fermentation broth in which the FK-506 is produced. Further, it can be used to select compounds which bind to the protein as a screening test for identifying new immunosuppressant drugs. In these various procedures, it is preferred, although not required, to immobilize the protein. This can be accomplished by any procedure known in the art. A particularly useful support for immobilizing proteins is cyanogen bromide treated Sepharose (CNBR-activated Sepharose 4B, Pharmacia, Piscataway, New Jersey). The immobilized protein is prepared by mixing the protein under basic conditions with the cyanogen bromide-activated Sepharose.

The binding of the protein to this matrix can result in a marked stabilization of the bound activity through a three-dimensional stabilization achieved by multiple bonds through the amino groups of the protein.

The preferred immobilized protein can also be used diagnostically for the determination of the concentration of FK-506 and its metabolites from physiological fluids, e.g. body fluids, and tissue extracts as for example in patients who are undergoing FK-506 immunosuppressive therapy.

The protein can also be used in an assay to bind FK-506 type macrolide compounds and biologically useful ligands, by allowing the protein and FK-506 type macrolide/ligand to form a complex in an excess of the macrolide, then eluding the mixture through a column and analyzing the concentration of the pure complex Spectrophotometrically or by scintillation counting. By this methodology, pure samples of the complex can be formed, from which the macrolide or biologically useful ligand can be isolated by interrupting the binding with an e.g.

strong ionic salt solution and followed by conventional chromatographic separation.

Various changes and modifications can be made in the products and processess of the present invention without departing from the spirit and scope thereof. The various embodiments which have been set forth herein and the following example are for the purpose of further illustrating the invention but are not intented to limit it Sequence Analysis - Sequence analysis of the FKBPs was performed using the programs of the GCG package described in Deveraux et al., Null. Acids Res., ,387-395 (1984).

The following are the sequence listings in the case: SEQUENCE LISTING (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1641 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA ( cDNA) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION:SEQ ID NO:1: GATTCCCCTC ACCCTCTGCA TCCTCAACCC CATCCAGTAC CTCGAAGTCC TCGAGCGGTG 60 GGACCCCGGC GGGTGAGGAG GAAGAGGAGG AGGAAGAGGA GGAAGAGGAT GAC C TGAGTG 120 AGCTGCCACC GCTGGAGGAC ATGGGACAAC CCCCGGCGGA GGAGGCTGAG CAGCCTGGGG 180 CCCTGGCCCG AGAGTTCCTT GCTGCCATGG AGCCCGAGCC CGCCCCAGCC CCGGCCCCAG 240 AAGAGTGGCT GGACATTCTG GGGAACGGGC TGTTGAGGAA GAAGACGCTG GTCCCAGGGC 300 CGCCAGGTTC GAGCCGCCCG GTCAAGGGCC AGGTGGTCAC CGTACATCTG CAGACGTCGC 360 TGGAGAATGG CACACGGGTG CAGGAGGAGC CGGAGC TGGT GTTCAC TCTG GGTGAC TGTG 420 ACGTCATCCA GGCCCTGGAT CTCAGTGTCC CACTCATGGA CGTGGGGGAG ACGGCCATGG 480 TCACTGCTGA CTCCAAGTAC TGCTACGGCC CCCAAGGCAG GAGCCCATAC ATCCCCCCGC 540 ACGCGGCCCT GTGCCTGGAG GTGACCCTGA AGACGGCTGT GGACGGGCCT GACCTGGAGA 600 TGCTCACGGG GCAGGAGCGC GTGGCCCTGG CCAACCGGAA GCGGGAGTGC GGCAACGCCC 660 ACTACCAGCG GGCGGACTTC GTCCTGGCCG CCAACTCCTA CGACCTCGCC ATCAAGGCTA 720 TCACCTCCAG CGCCAAAGTG GACATGACGT TCGAGGAGGA GGCACAGCTC CTGCAGTTGA 780 AGGTGAAGTG TCTGAACAAC CTGGCGGCCT CGCAGCTGAA GCTCGACCAC TACCGCGCAG 840 CCCTGCGCTC CTGCAGCCTT GTGCTGGAGC ACCAGCCAGA CAACATCAAG GCTCTCTTCC 900 GCAAGGGCAA GGTGCTGGCC CAGCAGGGGG AGTACAGTGA GGCCATCCCC ATCCTGAGGG 960 CAGCCCTGAA GCTGGAACCT TCCAACAAGA CGATCCACGC AGAGCTCTCA AAGCTGGTGA 1020 AGAAGCATGC GGCGCAGCGG AGCACGGAGA CCGCCTTGTA CCGGAAAATG CTGGGCAACC 1080 CCAGCCGGCT GCCTGCTAAG TGCCCTGGCA AGGGTGCCTG GTCCATCCCA TGGAAGTGGC 1140 TGTTTGGGGC GACTGCTGTT GCCTGGGGGG GTGTGGCACT CTCTGTGGTC ATCGCTGCCA 1200 GGAACTGACC ACCTAGGTGG CTGCCACCCC CTCTGCACAC CATGGACCCT GCCCTGCGCT 1260 CCCCAACTCC CCCAGGCTCC CTGTCCACTG CCCTCCCTGG TCTGGCCCCC TCCTCCGGGT 1320 TAGGGGAGCA AGGATTGGGG GTCGTGCAGC CCAGCCAGCA GGAGGGACTG AGGCCCTCTA 1380 GGAGGAAAGC CCAGAGGGAG GGGGCCCTCA TTCCTTCAGA CCCAGTTTTC CCCCACCCTC 1440 CTTACCCCGC TGGGCTAGGT CTCCGCCAGG GCTGGCCTCA GITTCTCCTC AACAGGCCTG 1500 GGGGCAGCCC TTCCCCTGCC TAGTCCCCGC CTGAGTGCCA GCCCCCCACC CCGCCTGCCG 1560 CCCCCTGTCC AGGTTCCCTC CCCGCCACAG TGAAATAAAG CATCCCACCC TGCAAAAAAA 1620 AAAAAAAAAA AAAAGGAATT C 1641 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 355 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION:SEQ ID NO:2: Met Gly Gln Pro Pro Ala Glu Glu Ala Glu Gln Pro Gly Ala Leu Ala 1 5 10 15 Arg Glu Phe Leu Ala Ala Met Glu Pro Glu Pro Ala Pro Ala Pro Ala 20 25 30 Pro Glu Glu Trp Leu Asp Ile Leu Gly Asn Gly Leu Leu Arg Lys Lys 35 40 45 Thr Leu Val Pro Gly Pro Pro Gly Ser Ser Arg Pro Val Lys Gly Gln 50 55 60 Val Val Thr Val His Leu Gln Thr Ser Leu Glu Asn Gly Thr Arg Val 65 70 75 80 Gln Glu Glu Pro Glu Leu Val Phe Thr Leu Gly Asp Cys Asp Val Ile 85 90 95 Gln Ala Leu Asp Leu Ser Val Pro Leu Met Asp Val Gly Glu Thr Ala 100 105 110 Met Val Thr Ala Asp Ser Lys Tyr Cys Tyr Gly Pro Gln Gly Arg Ser 115 120 125 Pro Tyr Ile Pro Pro His Ala Ala Leu Cys Leu Glu Val Thr Leu Lys 130 135 140 Thr Ala Val Asp Gly Pro Asp Leu Glu Met Leu Thr Gly Gln Glu Arg 145 150 155 160 Val Ala Leu Ala Asn Arg Lys Arg Glu Cys Gly Asn Ala His Tyr Gln 165 170 175 Arg Ala Asp Phe Val Leu Ala Ala Asn Ser Tyr Asp Leu Ala Ile Lys 180 185 190 Ala Ile Thr Ser Ser Ala Lys Val Asp Met Thr Phe Glu Glu Glu Ala 195 200 205 Gln Leu Leu Gln Leu Lys Val Lys Cys Leu Asn Asn Leu Ala Ala Ser 210 215 220 Gln Leu Lys Leu Asp His Tyr Arg Ala Ala Leu Arg Ser Cys Ser Leu 225 230 235 240 Val Leu Glu His Gln Pro Asp Asn Ile Lys Ala Leu Phe Arg Lys Gly 245 250 255 Lys Val Leu Ala Gln Gln Gly Glu Tyr Ser Glu Ala Ile Pro Ile Leu 260 265 270 Arg Ala Ala Leu Lys Leu Glu Pro Ser Asn Lys Thr Ile His Ala Glu 275 280 285 Leu Ser Lys Leu Val Lys Lys His Ala Ala Gln Arg Ser Thr Glu Thr 290 295 300 Ala Leu Tyr Arg Lys Met Leu Gly Asn Pro Ser Arg Leu Pro Ala Lys 305 310 315 320 Cys Pro Gly Lys Gly Ala Trp Ser Ile Pro Trp Lys Trp Leu Phe Gly 325 330 335 Ala Thr Ala Val Ala Leu Gly Gly Val Ala Leu Ser Val Val Ile Ala 340 345 350 Ala Arg Asn 355 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (cDNA) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 17 (C) OTHER INFORMATION: /label= N /note= ginosine (ix) FEATURE: (A) NAME/KEY:Modified-site (B) LOCATION: 20 (C) OTHER INFORMATION: /label= N /note= ZinosineZ (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 23 (C) OTHER INFORMATION: /label= N /note= ZinosineZ (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 26 -(C) OTHER INFORMATION: /label= N /note= Ninosine N (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 29 (C) OTHER INFORMATION: /label= N /note= Sinosinew (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: GGAATTCCTG GTAYACNGGN AHNYTNGANR AYGG 34 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (cDNA) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE:NO (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 17 (C) OTHER INFORMATION: /label= N /note= Ninosine N (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 20 (C) OTHER INFORMATION: /label= N /note= NinosineN (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 23 (C) OTHER INFORMATION: /label= N /note= NinosineN (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 26 (C) OTHER INFORMATION: /label= N /note= NinosineN (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 29 (C) OTHER INFORMATION: /label= N /note= Ninosine N (xi) SEQUENCE DESCRIPTION:SEQ ID NO:4: GGAATTCCCA YTAYACNGGN AHNYTNGANR AYGG 34 (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (CDNA) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 11 (C) OTHER INFORMATION: label N /note= Zinosinez (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 14 (C) OTHER INFORMATION: /label= N /note= "inosine" (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 17 (C) OTHER INFORMATION: /label= N /note= Ninosine N (ix) FEATURE: (A) NAME/KEY:Modified-site (B) LOCATION: 20 (C) OTHER INFORMATION: /label= N /note= NinosineN (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 26 (C) OTHER INFORMATION: /label= N /note= Ninosine N (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 29 (C) OTHER INFORMATION: /label= N /note= NinosineN (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 32 (C) OTHER INFORMATION: /label= N /note= Ninosine N (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: GGAATTCCGG NAHNYTNGAN GAYGGNAMNR WNTTYGA 37 (2) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (cDNA) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 11 (C) OTHER INFORMATION: /label= N /note= Winosinew (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 14 (C) OTHER INFORMATION: /label= N /note= NinosineN (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 17 (C) OTHER INFORMATION: /label= N /note= Zinosine (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 20 (C) OTHER INFORMATION: /label= N /note= NinosineN (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 26 (C) OTHER INFORMATION: /label= N /note= NinosineN (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 32 (C) OTHER INFORMATION: /label= N /note= inosineZ (xi) SEQUENCE DESCRIPTION:SEQ ID NO:6: GGAATTCCGG NAHNYTNGAN GAYGGNCARR WNTTYGA 37 (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (cDNA) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 11 (C) OTHER INFORMATION: /label= N /note= inosineZ (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 17 (C) OTHER INFORMATION: /label= N /note= Ninosine N (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 20 (C) OTHER INFORMATION: /label= N /note= NinosineN (ix) FEATURE: (A) NAME/KEY:Modified-site (B) LOCATION: 23 (C) OTHER INFORMATION: /label= N /note= SinosineZ (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 26 (C) OTHER INFORMATION: /label= N /note= inosine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: GGAATTCCSR NVARGTNATN MRNGGNTGG 29 (2) INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 bases (B) TYPE: nucleic acid -(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (CDNA) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 11 (C) OTHER INFORMATION: /label= N /note= Zinosinew (ix) FEATURE: (A) NAME/KEY:Modified-site (B) LOCATION: 17 (C) OTHER INFORMATION: /label= N /note= Ninosine N (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 20 (C) OTHER INFORMATION: /label= N /note= Ninosine N (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 23 (C) OTHER INFORMATION: /label= N /note= "inosine" (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 26 (C) OTHER INFORMATION: /label= N /note= "inosine" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: GGAATTCCSR NVARGTNATN MRNGGNTGGG A 31 (2) INFORMATION FOR SEQ ID NO:9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (cDNA) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE:YES (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 9 (C) OTHER INFORMATION: /label= N /note= "inosine N (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 12 (C) OTHER INFORMATION: /label= N /note= ZinosineX (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 15 (C) OTHER INFORMATION: /label= N /note= "inosine N (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 18 (C) OTHER INFORMATION: /label= N /note= "inosine" (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 21 (C) OTHER INFORMATION: /label= N /note= inosinew (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 27 (C) OTHER INFORMATION: /label= N /note= Winosinei (xi) SEQUENCE DESCRIPTION:SEQ ID NO:9: GGAATTCCNR SNABNSCNWS NTCCCANCC 29 (2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (cDNA) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (ix) FEATURE: (A) NAME/KEY: Modified-site -(B) LOCATION: 9 (C) OTHER INFORMATION: /label= N /note= "inosine" (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 12 (C) OTHER INFORMATION: /label= N /note= inosineS (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 15 (C) OTHER INFORMATION: /label= N /note= "inosine" (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 21 (C) OTHER INFORMATION: /label= N /note= "inosine" (ix) FEATURE: (A) NAME/KEY:Modified-site (B) LOCATION: 24 (C) OTHER INFORMATION: /label= N /note= "inosine" (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 27 (C) OTHER INFORMATION: /label= N /note= "inosine* (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 30 (C) OTHER INFORMATION: /label= N /note= inosinew (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: GGAATTCCNS CNWSNTCCCA NCCNKKNATN AC 32 (2) INFORMATION FOR SEQ ID NO:11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (cDNA) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (ix) FEATURE: (A) NAME/KEY:Modified-site (B) LOCATION: 11 (C) OTHER INFORMATION: /label= N /note= "inosine N (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 14 (C) OTHER INFORMATION: /label= N /note= "inosine N (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 17 (C) OTHER INFORMATION: /label= N /note= Sinosinei (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 20 (C) OTHER INFORMATION: /label= N /note= "inosine" (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 23 (C) OTHER INFORMATION: /label= N /note= "inosine" (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 26 (C) OTHER INFORMATION: /label= N /note= "inosine" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: GGAATTCCAR NKTNGMNYBN GSNGGNAT 28 (2) INFORMATION FOR SEQ ID NO:12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (cDNA) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 11 (C) OTHER INFORMATION: /label= N /note= "inosine" (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 14 (C) OTHER INFORMATION: /label= N /note= "inosine" (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 17 (C) OTHER INFORMATION: /label= N /note= inosinew (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 20 (C) OTHER INFORMATION: /label= N /note= NinosineN (ix) FEATURE: (A) NAME/KEY:Modified-site (B) LOCATION: 23 (C) OTHER INFORMATION: /label= N /note= "inosine" (A) NAME/KEY: Modified-site (B) LOCATION: 26 (C) OTHER INFORMATION: /label= N /note= ZinosineZ (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: GGAATTCCAR NKTNGMNYBN CCNGGNAT 28 (2) INFORMATION FOR SEQ ID NO:13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (CDNA) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 9 (C) OTHER INFORMATION: /label= N /note= "inosine" (ix) FEATURE: -(A) NAME/KEY: Modified-site (B) LOCATION: 15 (C) OTHER INFORMATION: /label= N /note= Zinosine (ix) FEATURE: (A) NAME/KEY:Modified-site (B) LOCATION: 18 (C) OTHER INFORMATION: /label= N /note= "inosine N (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 24 (C) OTHER INFORMATION: /label= N /note= "inosine N (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 27 (C) OTHER INFORMATION: /label= N /note= "inosine" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: GGAATTCCNA RYTCNRYNTC RAANRYNAG 29 (2) INFORMATION FOR SEQ ID NO:14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (cDNA) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 9 (C) OTHER INFORMATION: /label= N /note= "inosine" (ix) FEATURE: (A) NAME/KEY:Modified-site (B) LOCATION: 15 (C) OTHER INFORMATION: /label= N /note= NinosineN (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 18 (C) OTHER INFORMATION: /label= N /note= inosineN (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 24 (C) OTHER INFORMATION: /label= N /note= "inosine" (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 27 (C) OTHER INFORMATION: /label= N /note= "inosine" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: GGAATTCCNA RYTCNRYNTC RAANRYNAA 29 (2) INFORMATION FOR SEQ ID NO:15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 165 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (CDNA) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE:NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: ACACGGGTGC AGGAGGAGCC GGAGCTGGTG TTCACTCTGG GTGACTGTGA CGTCATCCAG 60 GCCCTGGATC TCAGTGTCCC ACTCATGGAC GTGGGGGAGA CGGCCATGGT CACTGCTGAC 120 TCCAAGTACT GCTACGGCCC CCAAGGCAGC AGGAGCCCAT ACATC 165 (2) INFORMATION FOR SEQ ID NO:16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 55 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: Thr Arg Val Gln Glu Glu Pro Glu Leu Val Phe Thr Leu Gly Asp Cys 1 5 10 15 Asp Val lie Gln Ala Leu Asp Leu Ser Val Pro Leu Met Asp Val Gly 20 25 30 Glu Thr Ala Met Val Thr Ala Asp Ser Lys Tyr Cys Tyr Gly Pro Gln 35 40 45 Gly Ser Arg Ser Pro Tyr Ile 50 55

Claims (9)

WHAT IS CLAIMED IS:

1. A DNA sequence coding for the human 3 8kDa FK-506 binding protein.

2. A DNA sequence as shown below coding for the human 38kDa FK-506 binding protein: (SEQ. ID No. 1) GATTCCCCTC ACCCTCTGCA TCCTCAACCC CATCCAGTAC CTCGAAGTCC TCGAGCGGTG 60 GGACCCCGGC GGGTGAGGAG GAAGAGGAGG AGGAAGAGGA GGAAGAGGAT GACCTGAGTG 120 AGCTGCCACC GCTGGAGGAC ATGGGACAAC CCCCGGCGGA GGAGGCTGAG CAGCCTGGGG 180 CCCTGGCCCG AGAGTTCCTT GCTGCCATGG AGCCCGAGCC CGCCCCAGCC CCGGCCCCAG 240 AAGAGTGGCT GGACATTCTG GGGAACGGGC TGTTGAGGAA GAAGACGCTG GTCCCAGGGC 300 CGCCAGGTTC GAGCCGCCCG GTCAAGGGCC AGGTGGTCAC CGTACATCTG CAGACGTCGC 360 TGGAGAATGG CACACGGGTG CAGGAGGAGC CGGAGCTGGT GTTCACTCTG GGTGACTGTG 420 ACGTCATCCA GGCCCTGGAT CTCAGTGTCC CACTCATGGA CGTGGGGGAG ACGGCCATGG 480 TCACTGCTGA CTCCAAGTAC TGCTACGGCC CCCAAGGCAG GAGCCCATAC ATCCCCCCGC 540 ACGCGGCCCT GTGCCTGGAG GTGACCCTGA AGACGGCTGT GGACGGGCCT GACCTGGAGA 600 TGCTCACGGG GCAGGAGCGC GTGGCCCTGG CCAACCGGAA GCGGGAGTGC GGCAACGCCC 660 ACTACCAGCG GGCGGACTTC GTCCTGGCCG CCAACTCCTA CGACCTCGCC ATCAAGGCTA 720 TCACCTCCAG CGCCAAAGTG GACATGACGT TCGAGGAGGA GGCACAGCTC CTGCAGTTGA 780 AGGTGAAGTG TCTGAACAAC CTGGCGGCCT CGCAGCTGAA GCTCGACCAC TACCGCGCAG 840 CCCTGCGCTC CTGCAGCCTT GTGCTGGAGC ACCAGCCAGA CAACATCAAG GCTCTCTTCC 900 GCAAGGGCAA GGTGCTGGCC CAGCAGGGGG AGTACAGTGA GGCCATCCCC ATCCTGAGGG 960 CAGCCCTGAA GCTGGAACCT TCCAACAAGA CGATCCACGC AGAGCTCTCA AAGCTGGTGA 1020 AGAAGCATGC GGCGCAGCGG AGCACGGAGA CCGCCTTGTA CCGGAAAATG CTGGGCAACC 1080 CCAGCCGGCT GCCTGCTAAG TGCCCTGGCA AGGGTGCCTG GTCCATCCCA TGGAAGTGGC 1140 TGTTTGGGGC GACTGCTGTT GCCTTGGGGG GTGTGGCACT CTCTGTGGTC ATCGCTGCCA 1200 GGAACTGACC ACCTAGGTGG CTGCCACCCC CTCTGCACAC CATGGACCCT GCCCTGCGCT 1260 CCCCAACTCC CCCAGGCTCC CTGTCCACTG CCCTCCCTGG TCTGGCCCCC TCCTCCGGGT 1320 TAGGGGAGCA AGGATTGGGG GTCGTGCAGC CCAGCCAGCA GGAGGGACTG AGGCCCTCTA 1380 GGAGGAAAGC CCAGAGGGAG GGGGCCCTCA TTCCTTCAGA CCCAGTTTTC CCCCACCCTC 1440 CTTACCCCGC TGGGCTAGGT CTCCGCCAGG GCTGGCCTCA GTTTCTCCTC AACAGGCCTG 1500 GGGGCAGCCC TTCCCCTGCC TAGTCCCCGC CTGAGTGCCA GCCCCCCACC CCGCCTGCCG 1560 CCCCCTGTCC AGGTTCCCTC CCCGCCACAG TGAAATAAAG CATCCCACCC TGCAAAAAAA 1620 AAAAAAAAAA AAAAGGAATT C , and variants thereof. 1641 -

3. The human 38 kDa FK-506 binding protein in substantially biologically pure form.

4. The human 38 kDa FK-506 binding protein having the amino acid sequence as shown below: (SEQ. ID No. 2) Met Gly Gln Pro Pro Ala Glu Glu Ala Glu Gln Pro Gly Ala Leu Ala 5 10 15 Arg Glu Phe Leu Ala Ala Met Glu Pro Glu Pro Ala Pro Ala Pro Ala 20 25 30 Pro Glu Glu Trp Leu Asp Ile Leu Gly Asn Gly Leu Leu Arg Lys Lys 35 40 45 Thr Leu Val Pro Gly Pro Pro Gly Ser Ser Arg Pro Val Lys Gly Gln 50 55 60 Val Val Thr Val His Leu Gln Thr Ser Leu Glu Asn Gly Thr Arg Val 65 70 75 80 Gln Glu Glu Pro Glu Leu Val Phe Thr Leu Gly Asp Cys Asp Val Ile 85 90 95 Gln Ala Leu Asp Leu Ser Val Pro Leu Met Asp Val Gly Glu Thr Ala 100 105 110 Met Val Thr Ala Asp Ser Lys Tyr Cys Tyr Gly Pro Gln Gly Arg Ser 115 120 125 Pro Tyr Ile Pro Pro His Ala Ala Leu Cys Leu Glu Val Thr Leu Lys 130 135 140 Thr Ala Val Asp Gly Pro Asp Leu Glu Met Leu Thr Gly Gln Glu Arg 145 150 155 160 Val Ala Leu Ala Asn Arg Lys Arg Glu Cys Gly Asn Ala His Tyr Gln 165 170 175 Arg Ala Asp Phe Val Leu Ala Ala Asn Ser Tyr Asp Leu Ala Ile Lys 180 185 190 Ala Ile Thr Ser Ser Ala Lys Val Asp Met Thr Phe Glu Glu Glu Ala 195 200 205 Gln Leu Leu Gln Leu Lys Val Lys Cys Leu Asn Asn Leu Ala Ala Ser 210 215 220 Gln Leu Lys Leu Asp His Tyr Arg Ala Ala Leu Arg Ser Cys Ser Leu 225 230 235 240 Val Leu Glu His Gln Pro Asp Asn Ile Lys Ala Leu Phe Arg Lys Gly 245 250 255 Lys Val Leu Ala Gln Gln Gly Glu Tyr Ser Glu Ala Ile Pro Ile Leu 260 265 270 Arg Ala Ala Leu Lys Leu Glu Pro Ser Asn Lys Thr Ile His Ala Glu 275 280 285 Leu Ser Lys Leu Val Lys Lys His Ala Ala Gln Arg Ser Thr Glu Thr 290 295 300 Ala Leu Tyr Arg Lys Met Leu Gly Asn Pro Ser Arg Leu Pro Ala Lys 305 310 315 320 Cys Pro Gly Lys Gly Ala Trp Ser Ile Pro Trp Lys Trp Leu Phe Gly 325 330 335 Ala Thr Ala Val Ala Leu Gly Gly Val Ala Leu Ser Val Val Ile Ala 340 345 350 Ala Arg Asn, and variants thereof.

355

5. A vector containing the DNA sequence of Claim 2.

6. A microbial host transformed by the vector of Claim 5 containing the DNA sequence coding for the human 38 kDa FK-506 binding protein.

7. A process for the preparation of the human 38 kDa FK-506 binding protein comprising culturing the transformed host of Claim 6 under conditions suitable for the expression of the human 38 kDa FK-506 binding protein, and recovering said binding protein.

8. Human 38 kDa FK-506 binding protein made by the process of Claim 7.

9. A method of testing chemical compounds in order to identify those which have immunosuppressive activity capable of blocking allograft rejection by preventing T-cell activation, comprising bringing said test chemical compound into contact with the human 38 kDa FK-506 binding protein and determining whether said test compound will bind thereto.