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Crystallized oxalate decarboxylase and methods of use

A technology of oxalate decarboxylase, oxalate, applied in the field of pharmaceutical composition, treatment of oxalate-related diseases such as hyperoxaluria, cross-linked oxalate decarboxylase crystal

Inactive Publication Date: 2009-11-18
ALTUS PHARMA INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These therapies (eg, low oxalate or low-fat diet, vitamin B6, adequate calcium, and increased fluids) are only partially effective, and they may have unwanted adverse side effects, such as orthophosphate, magnesium, or cholestene Gastrointestinal effects of amine supplementation, and risks of dialysis and surgery

Method used

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  • Crystallized oxalate decarboxylase and methods of use
  • Crystallized oxalate decarboxylase and methods of use
  • Crystallized oxalate decarboxylase and methods of use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0133] Example 1. Fermentation and purification of oxalate decarboxylase.

[0134] Oxalate decarboxylase (OXDC) from Bacillus subtilis (B. subtilis) is a 261 kDa homohexameric protein composed of 6 identical monomers. Each monomer contains 385 amino acids with a calculated molecular weight of -43-44 kDa and an isoelectric point of 5.2. The OXDC gene formerly known as yvrK was PCR amplified using Bacillus subtilis genomic DNA as a template.

[0135] The amplified OXDC gene was first cloned into the pCRII vector (Invitrogen, Carlsbad, CA), then subcloned, and expressed from the pET-11a expression vector using E. coli BL21(DE3) pLysS cells. This gene expression in the pET-11a vector is under the control of the T7 promoter, induced by IPTG (isopropyl-β-D-thiogalactoside), which regulates OXDC expression.

[0136] Fermentation was used to achieve high level expression of recombinant OXDC in E. coli. In the presence of casein hydrolyzate (USB Corporation, Cleveland, OH) or soy ...

Embodiment 2

[0140] Example 2. Using mild denaturant concentrations and subsequent anion exchange chromatography from solubilized Granular crystalline oxalate decarboxylase.

[0141] OXDC pellets refrigerated at -20°C were used to prepare OXDC crystals.

[0142] In this procedure, particles are dissolved under mild conditions of denaturant concentration and pH. The solubilized protein is then refolded using an anion exchange matrix column.

[0143] The particles were dissolved in 2M urea, 100 mM Tris pH 10.0, 10 mM DTT and 100 mM NaCl (1:10 w / v). The solution was stirred at room temperature (RT) for 2 h, then centrifuged at 15K for 30 min at 4°C. The supernatant was carefully decanted and stored. The pellets were carefully weighed and stored separately.

[0144] Under constant and gentle agitation, at a flow rate of 10 ml / min, the solubilized particles in the supernatant were added dropwise to 10 volumes of a solution consisting of 2M urea, 100 mM Tris pH 8.0, 1 mM DTT, and 1 mM Mn...

Embodiment 3

[0148] Example 3. Using high denaturant concentrations and subsequent anion exchange chromatography, by dissolution The particles crystallize oxalate decarboxylase.

[0149] By this method, particles were dissolved in 5M urea, 50 mM Tris pH 8.6, 100 mM NaCl, 10 mM DTT (1 :5 w / v). The solution was stirred at room temperature for 2 h and then centrifuged at 15K for 30 min at 4°C. The supernatant was carefully decanted and stored. The pellets were carefully weighed and stored separately.

[0150] Prepare anion exchange chromatography columns by packing Q Sepharose matrix into glass columns. The column was attached to the FPLC and equilibrated by washing with 3 column volumes (CV) of 4M urea, 100 mM Tris pH 8.6 and 10 mM DTT. With 7 column volumes of 100 mM NaCl, 50 mM Tris pH 8, 1 mM MnCl 2 , 10mM DTT, further wash the column with 3 column volumes of 0.5M NaCl, 50mM Tris pH 8.0, 1mM DTT, 1mM MnCl in a single step 2 elute.

[0151] Appropriate fractions were collected an...

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Abstract

Oxalate decarboxylase crystals, including stabilized crystals, such as cross-linked crystals of oxalate decarboxylase, are disclosed. Methods to treat a disorder associated with elevated oxalate concentration using oxalate decarboxylase crystals are also disclosed. Additionally disclosed are methods of producing protein crystals.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to US Application Serial No. 60 / 834,933, filed August 2, 2006, and US Application Serial No. 60 / 854,540, filed October 26, 2006, the contents of which are hereby incorporated by reference in their entirety. Background technique [0003] Oxalic acid is of the formula HO 2 C-CO 2 H dicarboxylic acid. Oxalic acid is mainly present in living organisms as oxalate, which is the salt form of oxalic acid. Oxalates are found in foods such as spinach, rhubarb, strawberries, cranberries, tree nuts, cocoa, chocolate, peanut butter, sorghum, and tea. Oxalate is also a metabolic end product in humans and other mammals. It is excreted in urine by the kidneys. When combined with calcium, oxalic acid produces an insoluble product, calcium oxalate, which is the most common compound found in kidney stones. [0004] Because mammals do not synthesize enzymes that degrade oxalate, oxalate levels in indiv...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/51
Inventor B·C·森诺伊T·G·卡彻罗J·施恩L·张A·拉实德D·格鲁季奇R·帕特尔M·E·麦克格拉斯
Owner ALTUS PHARMA INC
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