Insulin degrading enzyme crystals
AssigneeHoffmann-La Roche Inc.
InventorsJoerg Benz, Dominique Burger, Martine Stihle, Ralf Thoma
Life of this patent
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Abstract
The present invention provides apo crystals and co-crystals of insulin-degrading enzyme (IDE) and their uses in drug development.
Claims4
An apo crystal of an insulin-degrading enzyme (IDE) polypeptide, wherein the crystal belongs to space group P21, and wherein further the IDE polypeptide comprises amino acids 43-1018 of SEQ ID NO:2, and wherein further the crystal has unit cell dimensions of a=78±3 Å, b=115±3 Å, c=124±3 Å, β=97±3°.
A co-crystal of an insulin-degrading enzyme (IDE) polypeptide and an allosteric Ligand, wherein
- a) the crystal belongs to space group P21 and has unit cell dimensions of a=78±3 Å, b=115±3 Å, c=123±3 Å, β=97±3°,
- b) the IDE polypeptide comprises amino acids 43-1018 of SEQ ID NO:2 and
- c) the allosteric Ligand is 1H-Indole-7-carboxylic acid (3-chloro-phenyl)-amide.
A method of crystallizing an insulin-degrading enzyme (IDE) polypeptide, the method comprising:
- a) providing an 11 mg/ml aqueous solution of the IDE polypeptide, wherein said IDE polypeptide is a polypeptide comprising a sequence having an identity to a polypeptide of SEQ ID NO:
- 2 of at least 95%, and
- b) growing crystals by vapor diffusion or microbath at 4° C. via mixing 1.5 ul of the IDE protein solution with 0.3 ul of a seed stock solution and 0.5 ul of a reservoir solution, wherein said buffered reservoir solution comprises 100 mM Tris/HCl pH 8.5, 200 mM ammonium acetate, 25% (w/v) PEG5000 MME and 10% ethylene glycol.
A method for co-crystallizing an insulin-degrading enzyme (IDE) polypeptide with an allosteric Ligand, the method comprising:
- a) providing an 12 mg/ml aqueous solution of the IDE polypeptide, wherein said IDE polypeptide is a polypeptide comprising a sequence having an identity to a polypeptide of SEQ ID NO:
- 2 of at least 95%, and
- b) adding a molar excess of the allosteric Ligand to the aqueous solution of the polypeptide, wherein the allosteric Ligand is 1H-Indole-7-carboxylic acid (3-chloro-phenyl)-amide and
- c) growing crystals by vapor diffusion or microbath at 4° C. using via mixing 1.5 ul of the IDE protein solution with 0.5 ul of a seed stock solution and 0.5 ul of a reservoir solution, wherein said buffered reservoir solution comprises 100 mM Bis-Tris/HCl pH 6.5, 200 mM ammonium acetate, 25% (w/v) PEG1500, 4% PEG 400 and 10% ethylene glycol.
Dependencies are inferred from the claim text.