Efficient secretory expression and purification method of recombinant urate oxygen oxidoreductase

A urate oxidase and urate oxidase protein technology, applied in the field of medical bioengineering, can solve the problems of poor cytoplasmic expression, difficult control of protease cleavage conditions, non-specific cleavage, etc., and achieve low product cost and high purification The effect of short cycle and simple operation

Inactive Publication Date: 2013-06-26
吉林修正药业新药开发有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When Escherichia coli expresses Aspergillus flavus uric acid oxidase, the expression effect of its cytoplasmic expression form is not good; although the fusion expression method can solve the problem of expression level, the subsequent protease cleavage conditions are not easy to control, and it also introduces production problems. Difficulties such as protease residues and non-specific cleavage

Method used

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  • Efficient secretory expression and purification method of recombinant urate oxygen oxidoreductase
  • Efficient secretory expression and purification method of recombinant urate oxygen oxidoreductase
  • Efficient secretory expression and purification method of recombinant urate oxygen oxidoreductase

Examples

Experimental program
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Effect test

Embodiment 1

[0021] Example 1 Construction of recombinant urate oxidase secretion expression plasmid pPLS-UOX (see figure 1 ).

[0022] 1. Design and synthesize the Escherichia coli alkaline phosphatase promoter (phoA promoter).

[0023] EcoRI and XbaI restriction enzyme cutting sites were introduced at the 5' end and 3' end of the sequence of the Escherichia coli alkaline phosphatase promoter (phoA promoter), respectively. The designed and synthesized oligonucleotide sequence is SEQ1.

[0024] 2. Design and synthesize the coding sequence of Escherichia coli LamB signal peptide (LamB signal peptide) plus recombinant urate oxidase.

[0025] The coding sequence of the natural Escherichia coli LamB signal peptide is:

[0026] ATGATGATTA CTCTGCGCAA ACTgCCTCTG GCGGTTGCCG TCGCAGCGGG CGTAATGTCT GCTCAGGCAA TGGCTGTTGA TTTC

[0027] According to the codon preference of Escherichia coli, taking into account the influence of the secondary structure of post-transcriptional mRNA and the hydrophobici...

Embodiment 2

[0032] Example 2 Construction of engineering strain PLS-UOX for secretion and expression of recombinant urate oxidase.

[0033] E. coli empty host W3110 was transformed with plasmid pPLS-UOX, and the transformants were inoculated in AP5 medium and cultured in shake flasks at 37°C. The expression results were identified according to SDS-PAGE electrophoresis ( image 3 ) preferably obtains engineering bacteria that secrete and express recombinant urate oxidase.

[0034] The composition of AP5 medium is:

[0035] 0.15% glucose

[0036] 1.1% Acid Hydrolyzed Casein

[0037] 0.06% yeast extract

[0038] 0.019% MgSO 4

[0039] 0.107% NH 4 Cl

[0040] 0.373% KCl

[0041] 0.12% NaCl

[0042] 0.12mol / L triethanolamine pH7.4.

Embodiment 3

[0043] Example 3 Separation and Purification of Recombinant Urate Oxidase

[0044] Recombinant urate oxidase cells are crushed by high-pressure homogenization, the pressure is 30-40MPa, the centrifugal force of the broken bacteria is not less than 8000g, and the centrifuged supernatant is collected; the Cond value of the supernatant protein liquid is lower than the Cond value of the cationic chromatography equilibrium liquid Carry out cation chromatography, collect the sample, and the electrophoresis identification result of the purification effect is as follows: Figure 4 As shown; the pH value of the sample for cationic chromatography is adjusted to be consistent with the pH value of the hydrophobic chromatography equilibrium solution, and the hydrophobic chromatography is carried out to collect the sample for the sample, and the purification effect electrophoresis identification result is as follows Figure 5 Shown; The sample Cond value of the hydrophobic chromatography is...

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Abstract

The invention discloses an efficient secretory expression and purification method of a recombinant urate oxygen oxidoreductase, and belongs to the field of medical and biological engineering. The technical scheme comprises the following steps of: expressing an escherichia coli operon of the recombinant urate oxygen oxidoreductase using the encoding sequence of a recombinant urate oxygen oxidoreductase which is artificially synthesized from the encoding sequence of an escherichia coli alkaline phosphatase promoter and the encoding sequence of an escherichia coli LamB signal peptide; cloning the operon to a plasmid vector pBR322 carrying the escherichia coli to obtain an expression plasmid; transforming a W3110 escherichia coli empty host using the expression plasmid containing the escherichia coli operon to obtain an engineering strain for the secretory expression of the recombinant urate oxygen oxidoreductase; and carrying out purification technologies of cation-exchange chromatography, hydrophobic chromatography, and anion-exchange chromatography on the recombinant urate oxygen oxidoreductase. The method disclosed by the invention has the beneficial effects that a good secretory expression effect can be achieved through the regulation and control on protein expression by the escherichia coli alkaline phosphatase promoter and induction of the secretory expression of the urate oxygen oxidoreductase by the optimized escherichia coli LamB signal peptide, and the purification method is simple and easy in implementation, short in protein purification period and high in protein yield.

Description

technical field [0001] The invention relates to the field of medical bioengineering, in particular to the high-efficiency secretion expression of recombinant urate oxidase and the chromatographic separation and purification of recombinant urate oxidase protein. Background technique [0002] Uric acid oxidase (urate oxygen oxidoreductase, EC 1. 7. 3. 3, referred to as UC) is an enzyme in the purine degradation metabolic pathway in organisms, which can catalyze the oxidation of uric acid to generate allantoin, carbon dioxide and hydrogen peroxide. Uric acid oxidase is found in many species, but there is a lack of biologically active urate oxidase in higher mammals (apes and humans), so uric acid is the final product of purine metabolism. [0003] Studies have found that uric acid levels are associated with gout, hypertension, vascular disease, diabetes, kidney disease and many clinical syndromes. Urate oxidase, as a key reagent for detecting uric acid concentration in blood, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N15/70C12N1/21C12N9/06C12R1/19
Inventor 张军张博学李庆春杜研鞠蒙蒙
Owner 吉林修正药业新药开发有限公司
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