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WO1991004321A1 - Receptor for fk-506 - Google Patents

Receptor for fk-506 Download PDF

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Publication number
WO1991004321A1
WO1991004321A1 PCT/US1990/005449 US9005449W WO9104321A1 WO 1991004321 A1 WO1991004321 A1 WO 1991004321A1 US 9005449 W US9005449 W US 9005449W WO 9104321 A1 WO9104321 A1 WO 9104321A1
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gly
protein
val
lys
thr
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PCT/US1990/005449
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French (fr)
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Stuart L. Schreiber
Matthew W. Harding
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President And Fellows Of Harvard College
Yale University
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Publication of WO1991004321A1 publication Critical patent/WO1991004321A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants

Definitions

  • the invention relates to a substantially pure protein exhibiting isomerase activity, specifically cis-trans peptidyl-propyl isomerase activity, and which binds specifically to macrocycle FK506, a macrocycle having potent immunosuppressive activity.
  • the proteins of the present invention can be obtained in substantially pure form from cytosol extracts of human spleen and of bovine thymus as well as from bovine kidney, human and murine liver, and EL4 cells by passing the extracts through an affinity column containing an FK506 affinity matrix, then eluting with FK506.
  • Fig. 1 shows the chemical structure of FK506 where X is oxygen and R is hydrogen
  • Fig. 2 shows the chemical structure of rapamycin.
  • This derivative was diluted in ethanol to lmg/ml and mixed with an equal volume of Affigel 10 (BioRad) for four hours at room temperature with O.IN sodium bicarbonate. Unbound drug was recovered by washing the Affigel complex with isopropanol. Unreacted groups were blocked by shaking for four hours with 200mM ethanolamine hydrochloride in O.IN sodium bicarbonate. TLC anaylsis of wash fractions indicated more than 85% coupling of FK506.
  • Calf thymus and human spleen tissue were separately homogenized (l:4w/v) in lO TRIS, pH 7.4, 120mM potassium chloride, 5m 2-mercaptoethanol and ImM phenylmethylsulfonyl fluoride.
  • Ho ogenates were centrifuged at 8,000G for 20 minutes, then at 21,000rpm for 45 minutes and supernatants were clarified by filtration through a 0.45 ⁇ m membrane. Filtrates were diluted to 2-3mg protein/ L and 45-50mL passed through 0.5mL FK506-Affigel columns prepared as described above.
  • the proteins were recovered by batch elution of the affinity complex with FK506 (500 ⁇ g/mL in absolute ethanol) in a total volume of lmL. Approximately 15-30 ⁇ g of binding protein were recovered in each elution The elution fraction was lyophilized, and the protein re ⁇ c.
  • the low molecular weight (10-20kd) fraction was recovered and dialyzed extensively against 5mM TRIS pH 6.9 and the resulting proteins were resolved on a Synchropak CM-300 HPLC column (4.6mm IDx 25cm) by isocratic elution with 5mM TRIS, pH 6.9, 5mM sodium chloride at lmL/minute.
  • the FK506 binding activity of resolved proteins was monitored by Sephadex LH-20 partition assay as described by Handschumacher et al,
  • N-terminal sequence analysis was carried out by transferring the binding proteins to PVDF membranes by the procedure of Matsudaira et al, J. Biol. Chem. Vol. 261, 10,035-10,038 (1987) and membrane pieces were loaded into the cartridge of an Applied Biosystems Model 477A Gas Phase Sequencer with on line PTH amino acid analysis. Initial couplings and repetitive yields of 207 pmol/89% and 21 pmol/87% were calculated for the bovine and human sequences respectively. The sequences thus determined, starting from the N-terminal end, were as follows: for the binding protein from human spleen,
  • the resultant amplified DNA was cloned into pBS(+) (from Stratagene) .
  • the cDNA sequence was determined by the Sanger dideoxy termination procedure using a template single-stranded DNA obtained by helper phage rescue.
  • the isomerase activity of human FK506 binding protein was assayed by the procedure of Fischer et al, Nature, Vol. 337, 476-478 (1989). Cis-trans isomerization of succinyl-Ala-Ala-Pro-Phe-4-nitroanilide (27uM final concentration) was measured in a coupled assay with chymotrypsin, which hydrolyzes the anilide bond in the trans (but not cis) rotamer of the alanyl-prolyl containing peptide. The test peptide was preincubated with or without 0.67nM FK 506 binding protein at 10°C and the reaction was initiated by addition of chymotrypsin (27 uM final concentration).
  • the human and bovine FK506 binding proteins were also electrotransferred by the procedure of Gershoni et al. Anal. Biochem. , Vol 144, 32-40 (1985) to nitrocellulose and blots were developed with affinity purified rabbit anti-cyclophilin IgG (l. ⁇ g/mL) and I protein A(2xl0 cpm/mL) .
  • the rabbit anti-cyclophilin IgG did not react with either bovine or human FK506 binding protein but did react with cyclophilins.

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Abstract

Binding proteins of both human and bovine origin for the immunosuppressant FK506 have isomerase activity which is inhibited by FK506 but not by cyclosporin a. The N-terminal sequence of both proteins is H2-N-Gly-Val-Gln-Val-Glu-Thr-Ile-Ser-Pro-Gly-Asp-Gly-Arg-Thr-Phe-Pro-Lys-Arg-Gly-Gln-Thr-Cys-Val-Val-His-Tyr-Thr-Gly-Met-Leu-Glu-Asp-Gly-Lys-Lys-Phe-Asp-Ser-Ser-Arg. The entire sequence of the human protein deduced from the cloned human gene contains in addition: Asp-Arg-Asn-Lys-Pro-Phe-Lys-Phe-Met-Leu-Gly-Lys-Gln-Glu-Val-Ile-Arg-Gly-Trp-Glu-Glu-Gly-Val-Ala-Gln-Met-Ser-Val-Gly-Gln-Arg-Ala-Lys-Leu-Thr-Ile-Ser-Pro-Asp-Tyr-Ala-Tyr-Gly-Ala-Thr-Gly-His-Pro-Gly-Ile-Ile-Pro-Pro-His-Ala-Thr-Leu-Val-Phe-Asp-Val-Glu-Leu-Leu-Lys-Leu-Glu.

Description

RECEPTOR FOR FK-506
This invention was made with government support and the federal government has certain rights in the invention.
The invention relates to a substantially pure protein exhibiting isomerase activity, specifically cis-trans peptidyl-propyl isomerase activity, and which binds specifically to macrocycle FK506, a macrocycle having potent immunosuppressive activity.
The compound FK506 (Tanaka et al, J. Am. Chem. Soc. , Vol. 109, 5031-5033 (1987); Kino et al, J^. Antibiotics, Vol. 40, 1249-1256 (1987)) has been demonstrated to have potent immuosuppressive activity at concentrations several hundred fold lower than cyclosporin A, a cyclic* peptide which has found widespread clinical usage in the prevention of graft rejection following bone marrow and organ transplantation. The mechanisms of immunosuppression mediated by FK506 and cyclosporin A appear to be remarkably similar, suggesting that these unrelated chemical structures act on a common receptor or on similar molecular targets, perhaps the cyclosporin A receptor cyclophilin which exhibits cis-trans peptidyl-prolyl isomerase activity. Despite the similarity in activity between FK506 and cyclosporin A, the binding proteins of the present invention do not cross-react with antisera to cyclosporin A. The isomerase activity of the compounds of the present invention is inhibited by their binding to FK506. The properties and characteristics of the compounds of the present invention make them useful in enzyme inhibitor assays and receptor binding assays particularly in screening for new compounds having immunosuppressive activity. The proteins of the present invention can be obtained in substantially pure form from cytosol extracts of human spleen and of bovine thymus as well as from bovine kidney, human and murine liver, and EL4 cells by passing the extracts through an affinity column containing an FK506 affinity matrix, then eluting with FK506.
In the drawing,
Fig. 1 shows the chemical structure of FK506 where X is oxygen and R is hydrogen, and Fig. 2 shows the chemical structure of rapamycin.
Examples:
The synthesis of the FK506 affinity reagent or matrix began with stereo selective reduction of the carbon-22 ketone of FK506 with L-SeleGtride as described by Coleman et al, Heterocvcles, Vol. 28, 157-161 (1989) to an α-hydroxyl, β-hydrogen. Selective acylation of the carbon 32 hydroxyl group was carried out by reacting with β-azidoproprionic acid chloride followed by chemo-selective reduction of the azide to amino with 1, 3-dimercaptopropane under the conditions described by Bayley et al, Tetrahedron Lett., Vol. 39, 3633-3634 (1978), generating a carbon-32 amino derivative of the reduced FK506. This derivative was diluted in ethanol to lmg/ml and mixed with an equal volume of Affigel 10 (BioRad) for four hours at room temperature with O.IN sodium bicarbonate. Unbound drug was recovered by washing the Affigel complex with isopropanol. Unreacted groups were blocked by shaking for four hours with 200mM ethanolamine hydrochloride in O.IN sodium bicarbonate. TLC anaylsis of wash fractions indicated more than 85% coupling of FK506. Calf thymus and human spleen tissue were separately homogenized (l:4w/v) in lO TRIS, pH 7.4, 120mM potassium chloride, 5m 2-mercaptoethanol and ImM phenylmethylsulfonyl fluoride. Ho ogenates were centrifuged at 8,000G for 20 minutes, then at 21,000rpm for 45 minutes and supernatants were clarified by filtration through a 0.45 μm membrane. Filtrates were diluted to 2-3mg protein/ L and 45-50mL passed through 0.5mL FK506-Affigel columns prepared as described above. After extensive washing with phosphate buffered saline-0.05% TWEEN 20 (50mL) and then with 5mM TRIS, pH 7.4, 5mM 2-mercaptoethanol (5mL), the proteins were recovered by batch elution of the affinity complex with FK506 (500 μg/mL in absolute ethanol) in a total volume of lmL. Approximately 15-30 μg of binding protein were recovered in each elution The elution fraction was lyophilized, and the protein reεc.'ιv**-d by reducing SDS-P^GE "(12.5% gels) and identified by silver staining using molecular weight markers: lysozyme (14,400), trypsin (21,500), carbonic anhydrase (31,000), ovalbumin (45,000), albumin (66,200) and phosphorylase (97,500). The relative molecular weight of the FK506 binding protein from both human spleen and bovine thymus was determined by this procedure to be approximately 14,000kd. The FK506 binding protein was also eluted from the affinity complex by rapamycin (Fig. 2) at 500mg/mL in absolute ethanol. The binding protein was not eluted from the affinity complex by cyclosporin A. For purification of FK506 binding protein entirely free from FK506, clarified tissue homogenates from human spleen and bovine thymus respectively were heated at 60°C for 20 minutes. Supernatants harvested after centrifugation at 20,000 r m were concentrated, dialyzed against 20m TRIS pH 7.4 and chromatographed on a DEAE Sepharose column (1.6cm IDx 40cm) equilibrated with dialysis buffer. The void fractions were recovered, concentrated, and molecular weight fractions separated on a Sephacryl S100 HR column (2.5cm IDx 90cm) that was equilibrated with 20mM TRIS, pH 7.4, 150mM sodium chloride. The low molecular weight (10-20kd) fraction was recovered and dialyzed extensively against 5mM TRIS pH 6.9 and the resulting proteins were resolved on a Synchropak CM-300 HPLC column (4.6mm IDx 25cm) by isocratic elution with 5mM TRIS, pH 6.9, 5mM sodium chloride at lmL/minute. The FK506 binding activity of resolved proteins was monitored by Sephadex LH-20 partition assay as described by Handschumacher et al,
Science Vol. 226, 544-546 (1984). The FK506 binding protein in each case was associated with a single peak with a retention time of seven minutes. This protocol achieved a recovery of approximately 2μg FK506 binding protein per milligram of protein in the original clarified tissue homogenates.
Homogeneity of human 'and bovine FK506 binding protein was verified by silver staining after SDS-PAGE, isoelectric focussing
(pi 8.8-8.9), and N-terminal sequence analysis. The N-terminal sequence analysis was carried out by transferring the binding proteins to PVDF membranes by the procedure of Matsudaira et al, J. Biol. Chem. Vol. 261, 10,035-10,038 (1987) and membrane pieces were loaded into the cartridge of an Applied Biosystems Model 477A Gas Phase Sequencer with on line PTH amino acid analysis. Initial couplings and repetitive yields of 207 pmol/89% and 21 pmol/87% were calculated for the bovine and human sequences respectively. The sequences thus determined, starting from the N-terminal end, were as follows: for the binding protein from human spleen,
-H2N-Gly-Val-Gln-Val-Glu-Thr-Ile-Ser-Pro-Gly-Asp-Gly-Arg-Thr-Ph e-Pro; for the binding protein from bovine thymus the first sixteen residues were the same as above and the next 24 residues in sequence were
-Lys-Arg-Gly-Gln-Thr-Cys-Val-Val-His-Tyr-Thr-Gly-Met-Leu- Glu-Asp-Gly-Lys-Lys-Phe-Asp-Ser-Ser-Arg. The entire amino acid sequence of the human FK506 binding protein was deduced from the cDNA sequence. of the human gene as follows. Total RNA from Jurkat cells was copied using reverse transcriptase. The resulting cDNA pool was screened with oligonucleotide primers that were designed for use in the RACE-PCR (Frohman, et al . Proc. Natl, Acad. Sci., 85,
8998-9002) protocol. The resultant amplified DNA was cloned into pBS(+) (from Stratagene) . The cDNA sequence was determined by the Sanger dideoxy termination procedure using a template single-stranded DNA obtained by helper phage rescue. From this analysis the entire amino-acid sequence for the human FK506 binding protein was determined to be: Gly-Val-Gln-Val-Glu-Thr-Ile-Ser-Pro-Gly-Asp-Gly-Arg-Thr- Phe-Pro-Lys-Arg-Gly-Gln-Thr-Cys-Val-Val-His-Tyr-Thr-Gly- Met-Leu-Glu-Asp-Gly-Lys-Lys-Phe-Asp-Ser-Ser-Arg-Asp-Arg- Asn-Lys-Pro-Phe-Lys-Phe-Met-Leu-Gly-Lys-Gln-Glu-Val-Ile- Arg-Gly-Trp-Glu-Glu-Gly-Val-Ala-Gln-Met-Ser-Val-Gly-Gln- Arg-Ala-Lys-Leu-Thr-Ile-Ser-Pro-Asp-Tyr-Ala-Tyr-Gly-Ala- Thr-Gly-His-Pro-Gly-Ile-Ile-Pro-Pro-His-Ala-Thr-Leu-Val- Phe-Asp-Val-Glu-Leu-Leu-Lys-Leu-Glu. Increasing amounts of bovine FK506 binding protein were mixed with a fixed amount of FK506 labeled at carbon 32 by replacing R with - C 14C] - benzoyl. Complexes of the FK506 bovine binding protein with the 4C labeled FK506 were recovered by a Sephadex LH-20 partition assay as described in Handschumacher et al, supra, showing a linear relationship between the amount of binding protein and the amount of complex. In addition, titration with unlabeled FK506 against the complex of FK506 binding protein and labeled FK506 was carried out, showing a linearly proportional displacement of labeled ligand from the complex. On the other hand, there was no binding or complex formation between cyclosporin A and the FK506 binding proteins.
The isomerase activity of human FK506 binding protein was assayed by the procedure of Fischer et al, Nature, Vol. 337, 476-478 (1989). Cis-trans isomerization of succinyl-Ala-Ala-Pro-Phe-4-nitroanilide (27uM final concentration) was measured in a coupled assay with chymotrypsin, which hydrolyzes the anilide bond in the trans (but not cis) rotamer of the alanyl-prolyl containing peptide. The test peptide was preincubated with or without 0.67nM FK 506 binding protein at 10°C and the reaction was initiated by addition of chymotrypsin (27 uM final concentration). Reactions were peςfortaed in 100 mM TRIS, pH 8.0 and the hydrolysis of the 4-nitroanilide in the trans-rotamer of the peptide substrate was monitored at 395 nm with a Kontron Uvicon 860 spectrophoto eter. FK506 at final concentrations of 27, 54 and 270 nM resulted in increasing levels of inhibition. Whereas 5μM FK506 resulted in complete inhibition of enzyme activity, 5μM cyclosporin A had no observable effect.
The human and bovine FK506 binding proteins were also electrotransferred by the procedure of Gershoni et al. Anal. Biochem. , Vol 144, 32-40 (1985) to nitrocellulose and blots were developed with affinity purified rabbit anti-cyclophilin IgG (l.μg/mL) and I protein A(2xl0 cpm/mL) . The rabbit anti-cyclophilin IgG did not react with either bovine or human FK506 binding protein but did react with cyclophilins. It also reacted with a different protein having a molecular weight of approximately 14kd and obtained from bovine thymus extract by adsorption with a cyclosporin A affinity matrix followed by elution with cyclosporin A. Fragments of the human and bovine FK506 binding proteins whose isolation and purification is described above can be made having at least as much isomerase activity as described above.

Claims

Claims
1. A substantially pure protein which binds specifically to macrocycle FK506, has a molecular weight of approximately 11,800-14,OOOkd, and a pi of 8.8 to 8.9.
2. A protein as claimed in claim 1 which has an N-terminal sequence in which the first 16 residues are in sequence H2N-Gly-Val-Gln-Val-Glu-Thr-Ile-Ser-Pro-Gly-Asp-Gly-Arg- Thr-Phe-Pro.
3. A protein as claimed in claim 1 or 2 which is of human origin.
4. A protein as claimed in claim 1 or 2 which is of bovine origin.
« 5. A protein as claimed in claim 2 in which the next 24 residues are in sequence -Lys-Arg-Gly-Gln-Thr-Cys-Val-Val-His-Tyr-Thr-Gly-Met-Leu- Glu-Asp-Gly-Lys-Lys-Phe-Asp-Ser-Ser-Arg.
6. A protein as claimed in claim 5 which is of bovine origin.
7. A protein claimed in claim 3 having the amino acid sequence Gly-Val-Gln-Val-Glu-Thr-Ile-Ser-Pro-Gl -Asp-Gly-Arg-Thr- Phe-Pro-Lys-Arg-Gly-Gln-Thr-Cys-Val-Val-His-Tyr-Thr-Gly- Met-Leu-Glu-Asp-Gly-Lys-Lys-Phe-Asp-Ser-Ser-Arg-Asp-Arg- Asn-Lys-Pro-Phe-Lys-Phe-Met-Leu-Gly-Lys-Gln-Glu-Val-Ile- 7 Arg-Gly-Trp-Glu-Glu-Gly-Val-Ala-Gln-Met-Ser-Val-Gly-Gln-
8 Arg-Ala-Lys-Leu-Thr-Ile-Ser-Pro-Asp-Tyr-Ala-Tyr-Gly-Ala-
9 Thr-Gly-His-Pro-Gly-I le-I le-Pro-Pro-His-Ala-Thr-Leu-Val- ) 10 Phe-Asp-Val-Glu-Leu-Leu-Lys-Leu-Glu .
1 8. A protein as claimed in any one of claims 1
2 through 6 which does not cross react with antisera to
3 cyclosporin A.
1 9. A protein as claimed in any one of claims 1
2 through 6 which exhibits isomerase activity.
1 10. Fragments of the protein of claim 1 having at
2 least as much isomerase activity as said protein.
1 11. Fragments of the protein of claim 2 having at
2 least as much isomerase activity as said protein.
1 12. Fragments of the protein of claim 3 having at
2 least as much isomerase activity as said protein.
1 13. Fragments of the protein of claim 4 having at
2 least as much isomerase activity as said protein.
1 14. Fragments of the protein of claim 5 having at
2 least as much isomerase activity as said protein.
1 15. Fragments of the protein of claim 6 having at
2 least as much isomerase activity as said protein.
1 16. Fragments of the protein of claim 7 having at
2 least as much isomerase activity as said protein.
17. Fragments as claimed in any one of claims 9 through 15 which do not cross react with antisera to cyclosporin A.
PCT/US1990/005449 1989-09-25 1990-09-25 Receptor for fk-506 WO1991004321A1 (en)

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Application Number Priority Date Filing Date Title
US41208889A 1989-09-25 1989-09-25
US412,088 1989-09-25
US46497890A 1990-01-16 1990-01-16
US464,978 1990-01-16

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0481673A2 (en) * 1990-10-15 1992-04-22 Merck & Co. Inc. Genes encoding the human FK-506 binding protein and a S. cerevisiae homolog, FKB1, and their expression
EP0482189A1 (en) * 1990-05-09 1992-04-29 Children's Research Institute Receptor assay for fk-506
WO1992019745A1 (en) * 1991-05-08 1992-11-12 Vertex Pharmaceuticals Incorporated Rfkbp: a novel prolyl isomerase and rapamycin/fk506 binding protein
EP0594847A4 (en) * 1990-07-11 1993-06-14 Tonen Corp DNA ENCODING A HUMAN FK506 BINDING PROTEIN AND EXPRESSION OF THIS DNA.
FR2688227A1 (en) * 1992-03-04 1993-09-10 Inst Nat Sante Rech Med PROTEINS FORMING COMPLEXES WITH CHAPERONES AND THEIR LIGANDS, THEIR FRAGMENTS, THEIR PRODUCTION AND THEIR BIOLOGICAL APPLICATIONS.
US5354845A (en) * 1993-04-21 1994-10-11 Children's Research Institute FK-506 and rapamycin specific binding immunophilin
US5457182A (en) * 1994-02-15 1995-10-10 Merck & Co., Inc. FK-506 cytosolic binding protein, FKBP12.6
US5928868A (en) * 1996-04-26 1999-07-27 Massachusetts Institute Of Technology Three hybrid screening assay
US6104995A (en) * 1996-08-30 2000-08-15 Fujitsu Limited Speaker identification system for authorizing a decision on an electronic document
US7642059B2 (en) 2005-09-07 2010-01-05 Roche Diagnostics Operations, Inc. Single receptor assays for immunosuppressive drugs

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
NATURE, Volume 341, issued 26 October 1989, HARDING et al., "A Receptor for the Immunosuppressant FK-506 is a Cis-Trans Peptidyl-Prolyl Isomerase", pp. 758-760. *
NATURE, Volume 341, issued 26 October 1989, SIEKIERKA et al., "A Cytosolic Binding Protein for the Immunosuppressant FK-506 has Peptidyl-Prolyl Isomerase Activity but is Distinct from Cyclophilin", pp. 755-757. *

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0482189A4 (en) * 1990-05-09 1994-03-30 Children's Research Institute
EP0482189A1 (en) * 1990-05-09 1992-04-29 Children's Research Institute Receptor assay for fk-506
EP0594847A4 (en) * 1990-07-11 1993-06-14 Tonen Corp DNA ENCODING A HUMAN FK506 BINDING PROTEIN AND EXPRESSION OF THIS DNA.
EP0594847A1 (en) * 1990-07-11 1994-05-04 Tonen Corporation Dna coding for human fk506-binding protein and expression thereof
EP0481673A2 (en) * 1990-10-15 1992-04-22 Merck & Co. Inc. Genes encoding the human FK-506 binding protein and a S. cerevisiae homolog, FKB1, and their expression
EP0481673A3 (en) * 1990-10-15 1992-11-19 Merck & Co. Inc. Genes encoding the human fk-506 binding protein and a s. cerevisiae homolog, fkb1, and their expression
WO1992019745A1 (en) * 1991-05-08 1992-11-12 Vertex Pharmaceuticals Incorporated Rfkbp: a novel prolyl isomerase and rapamycin/fk506 binding protein
WO1993018146A2 (en) * 1992-03-04 1993-09-16 Institut National De La Sante Et De La Recherche Medicale (I.N.S.E.R.M.) Proteins forming complexes with chaperones and ligands thereof, fragments thereof, preparation thereof and biological uses thereof
WO1993018146A3 (en) * 1992-03-04 1993-11-11 Inst Nat Sante Rech Med Proteins forming complexes with chaperones and ligands thereof, fragments thereof, preparation thereof and biological uses thereof
FR2688227A1 (en) * 1992-03-04 1993-09-10 Inst Nat Sante Rech Med PROTEINS FORMING COMPLEXES WITH CHAPERONES AND THEIR LIGANDS, THEIR FRAGMENTS, THEIR PRODUCTION AND THEIR BIOLOGICAL APPLICATIONS.
US5354845A (en) * 1993-04-21 1994-10-11 Children's Research Institute FK-506 and rapamycin specific binding immunophilin
WO1994024165A1 (en) * 1993-04-21 1994-10-27 Children's Research Institute An fk-506 and rapamycin specific binding immunophilin
US5525523A (en) * 1993-04-21 1996-06-11 Children's Research Institute Binding method for FK-506- and rapamycin-like drugs with a novel immunophilin
US5457182A (en) * 1994-02-15 1995-10-10 Merck & Co., Inc. FK-506 cytosolic binding protein, FKBP12.6
US5928868A (en) * 1996-04-26 1999-07-27 Massachusetts Institute Of Technology Three hybrid screening assay
US6104995A (en) * 1996-08-30 2000-08-15 Fujitsu Limited Speaker identification system for authorizing a decision on an electronic document
US7642059B2 (en) 2005-09-07 2010-01-05 Roche Diagnostics Operations, Inc. Single receptor assays for immunosuppressive drugs

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AL Designated countries for regional patents

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