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Method for purifying recombinant aspergillus flavus uricase expressed by bacillus coli

A technology of uric acid oxidase and Escherichia coli, applied in the field of protein separation and purification, can solve the problems of increasing purification cost and production cycle, expensive protease price, long enzyme digestion time, etc., and achieves easy automation, low cost and short production cycle. Effect

Active Publication Date: 2011-11-23
HUBEI UNIV OF TECH +1
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  • Description
  • Claims
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Problems solved by technology

This method utilizes the N-terminal fusion tag to assist the purification of the target protein, which has high efficiency. However, after purification, the target protein needs to be digested and purified again to finally obtain a product with application value, and the protease used is relatively expensive. The cutting time is longer (overnight), which significantly increases the purification cost and production cycle
In addition, Chen Wei et al. also introduced a method of expressing Aspergillus flavus uricase in Escherichia coli using codon technology in their invention patent "A Method for Expressing Aspergillus flavus Urate Oxidase and a Special Gene", and the expression level of this method is relatively high (the expression level is about 40%), but its purification steps are relatively many and the cycle is long

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  • Method for purifying recombinant aspergillus flavus uricase expressed by bacillus coli

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Embodiment 1

[0057] A method for isolating and purifying recombinant Aspergillus flavus uricase expressed by Escherichia coli, the steps are:

[0058] A. Cloning of Aspergillus flavus urate oxidase cDNA:

[0059] Aspergillus flavus was cultivated for 3 days, the bacterial liquid was filtered, and the mycelium was collected. The total RNA of Aspergillus flavus mycelium was extracted according to the instructions of the Invitrogen Trizol RNA kit, and the total RNA of Aspergillus flavus mycelium was extracted with UricaseP1 (5-GGAATTCCATATGTCCGCAGTAAAAGCAGCCCGCTACG-3) and UricaseP2 (5-AGCCTCGAGTTATTACAATTTAGACTTCAGAGAGGACCG- 3) As primers, perform RT-PCR amplification according to the operating instructions of the RT-PCR kit of Promega Company. The amplified product (about 900bp) was connected to the T cloning vector pMD18-T of Takara Company, and Escherichia coli DH5α (purchased from Sangon Biotech (Shanghai) Co., Ltd.) was transformed by electroporation, and white colonies were picked for i...

Embodiment 2

[0079] In vitro biological activity assay of recombinant Aspergillus flavus uric acid oxidase:

[0080] Add 3 mL of 100 μmol / L uric acid solution dissolved in 3 mL of TEA buffer solution (7.5 g / L triethanolamine, 0.38 g / L EDTA, pH 8.9) into a 4 mL cuvette, then add 1 μg of purified urate oxidase, at 25 °C Add 0.5 mL of 20% (mass volume ratio) KOH to terminate the reaction after reacting for 5 min under the condition. The activity of the enzyme is obtained by detecting the decrease of the concentration of uric acid reflected by the decrease of the absorbance value at 292nm. Enzyme activity definition: under the conditions of pH 8.9 and 30°C, the amount of enzyme required to catalyze the oxidation of 1 μmol of uric acid per minute is one unit. Finally, the specific activity of the purified Aspergillus flavus uricase was measured to be 30 IU / mg.

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Abstract

The invention discloses a method for extracting recombinant aspergillus flavus uricase from an efficiently-expressed genetic engineering bacterium. The bacterium is bacillus coli rosetta (DE3) carrying a recombinant plasmid, and the recombinant plasmid is a plasmid pET-30c(+) containing an aspergillus flavus uricase gene. A method for producing the aspergillus flavus uricase by using the bacterium comprises the following steps of: A, fermenting the engineering bacterium and accumulating intracellular uricase; B, ultrasonically performing cell disruption and centrifugally collecting supernatant (containing soluble uricase); C, performing ion exchange chromatography, hydrophobic chromatography and gel permeation chromatography in sequence and collecting an activity peak; D, performing freeze drying on the activity peak to obtain the high-purity recombinant aspergillus flavus uricase. The method has the advantages of low production cost of the recombinant aspergillus flavus uricase, high enzyme expression level, simple and convenient purifying steps, high yield, high purity, easiness for industrial mass production and high practical application value.

Description

technical field [0001] The present invention belongs to the field of protein separation and purification, and in particular relates to a method for separating and purifying recombinant Aspergillus flavus uric acid oxidase expressed by Escherichia coli. Background technique [0002] Uric acid (also known as 2, 6, 8--trioxypurine) is the final product of the metabolism of purine compounds in the human body. The blood uric acid level depends on the dynamic balance of uric acid production and excretion, and the saturation of plasma uric acid: 37 ° C, PH7 .4: 380-420 μmol / L (6.4-7.1 mg / dL). [0003] In normal human blood circulation, 99% of uric acid exists in the form of sodium uric acid, and serum uric acid fluctuates within a narrow range. According to domestic data, the average male is 5.7mg / dL, and the female is 4.3mg / dL. If the serum uric acid is Concentrations higher than the normal value lead to hyperuricemia (Hyperuricemia). The proliferation of malignant tumors will a...

Claims

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Application Information

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IPC IPC(8): C12N9/06C12N15/70C12R1/19C12R1/67
Inventor 胡征杨波刘志刚董俊操志斐
Owner HUBEI UNIV OF TECH
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