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PEG-modified uricase

A uricase and carbamate-based technology, applied in the fields of enzymes, enzyme stability, urinary system diseases, etc.

Inactive Publication Date: 2005-01-05
SHENYANG SUNSHINE PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The clinical use of uricase is also limited by its short circulating half-life

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1: Isolation of Candida utilis Uricase Coding Sequence and Construction of Expression Plasmid

[0070] Genomic DNA was isolated from Candida utilis (ATCC 9950) and used as a PCR template to isolate the uricase gene. Candida utilis was grown on a shaker at 30℃×250rpm in 100mL YPD medium. The next day, cells from a 50 mL culture were pelleted by centrifugation at 1500 xg for 10 minutes at room temperature. The pellet was resuspended in 15 mL SCED buffer, pH 7.5 (1M sorbitol, 10 mM sodium citrate, pH 7.5, 10 mM EDTA, 10 mM DTT). 3mg Lyticas TM (Sigma, St. Louis, MO, catalog number L-4025) cells were added and cells were incubated at 37°C for 60 minutes. Add 15 mL of 1% SDS, mix slowly and place on ice for 5 minutes. Add 6 mL of 5M potassium acetate, pH 8.9 and mix slowly. The solution was centrifuged at 10,000xg for 10 minutes at 4°C. Transfer the supernatant to a clean centrifuge tube, add 2 volumes of ethanol and incubate at room temperature for 15 minutes. The DNA w...

Embodiment 2

[0075] Example 2: Expression of Uricase in Escherichia coli

[0076] To produce uricase, PHX12 is grown in 20L fermentation broth. E. coli undetermined medium #1 was used for PHX12 growth in a Bioflo benchtop fermentor (New Brunswick Scientific, Edison, NJ). The composition of E. coli undetermined medium #1 consists of basic medium, 50% glycerol, 100X salt solution, 100X calcium chloride solution and 1000X vitamin solution. These ingredients are prepared as described below.

[0077] Basic medium

[0078] Per liter of medium

[0079] Casamino acid 30g

[0080] Ammonium sulfate 3g

[0081] Potassium phosphate, dibasic value 2.5g

[0082] Dissolve in 920mL water and autoclave or filter sterilize through 0.22μm filter.

[0083] Concentrated salt solution (100X)

[0084] Boric acid 0.57g

[0085] Copper(II) sulfate pentahydrate 0.39g

[0086] Ferric chloride, 100g in 40mL water 2.0ml

[0087] Manganese dichloride tetrahydrate 4.0g

[0088] Sodium chloride 5.0g

[0089] Sodium molybdate...

Embodiment 3

[0112] Example 3: Purification of Uricase

[0113] The cell pellet from 20 liters of fermentation broth was resuspended in 0.4 L of lysis buffer (20 mM sodium phosphate, pH 8.5, 1 mM EDTA), using a PolytronTM homogenizer to obtain a uniform suspension. Cells were lysed by passing through a microfluidizer twice at >15,000 psi. The lysed cell suspension was then centrifuged at 13,000xg for 10 minutes. Ammonium sulfate was added to the supernatant to reach 30% saturation. The suspension was stirred at room temperature for 10 minutes, and then centrifuged at 13,000 xg for 15 minutes. Ammonium sulfate was added to the supernatant to a saturation of 64% and the solution was stirred at room temperature for 10 minutes, then the solution was centrifuged at 13,000×g for 15 minutes. The pellet was resuspended in 0.4 L of diafiltration buffer (20 mM sodium phosphate buffer, pH 8.5) and diafiltered against 5 volumes of diafiltration buffer with a 50,000 MW cutoff filter. The diafiltration solu...

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PUM

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Abstract

The present invention is directed to uricase modified with polyethylene glycol and to methods of treating different illnesses characterized by increased circulating uric acid levels, including but not limited to, hyperuricemia and tumor lysis syndrome.

Description

Field of invention [0001] The present invention is directed to polyethylene glycol-modified uricase and methods for treating different diseases characterized by increased circulating uric acid levels. Background of the invention [0002] Uric acid is a product of purine metabolism in birds, reptiles and primates, including humans. Uric acid is an intermediate in the catabolism of guanine nucleotides, and hypoxanthine is produced during the decomposition of adenine nucleotides. Uric acid is produced in the liver through the oxidation of xanthine and hypoxanthine. In most mammals, uric acid is further oxidized to allantoin by the enzyme urate oxidase. Due to the loss of its pyrimidine ring, allantoin exhibits water solubility that is more than 20 times that of uric acid. [0003] Uric acid oxidase, also called uricase, is an enzyme of the purine degradation pathway. Uricase catalyzes uric acid + O 2 Converted to allantoin + CO 2 . [0004] Humans lack uricase and cannot produce all...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09A61K38/00A61K38/44A61K47/48A61P13/00A61P13/12A61P19/06A61P35/00C12N9/06
CPCA61K38/00C12N9/0046A61P13/00A61P13/12A61P19/06A61P35/00
Inventor C·M·恩索M·A·克拉克F·W·霍尔茨伯格
Owner SHENYANG SUNSHINE PHARMA CO LTD
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