A kind of preparation method and application of soluble oxalate oxidase

An oxalate oxidase and soluble technology, applied in biochemical equipment and methods, oxidoreductase, and microbial-based methods, can solve the problems of cumbersome, high-cost, and difficult-to-achieve high-throughput assays for the determination of oxalate decarboxylase activity , to achieve the effect of easy high-throughput measurement, low cost and short time consumption

Active Publication Date: 2020-02-21
WUHAN KANGFUDE BIOTECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In order to solve the problems in the prior art that it is difficult to achieve soluble recombinant expression of oxalate oxidase in Escherichia coli and the determination method of oxalate decarboxylase activity is cumbersome, high cost and difficult to achieve high-throughput determination, the present invention provides a meth

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  • A kind of preparation method and application of soluble oxalate oxidase
  • A kind of preparation method and application of soluble oxalate oxidase
  • A kind of preparation method and application of soluble oxalate oxidase

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Experimental program
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Effect test

Embodiment 1

[0044] Embodiment 1 Obtaining of oxalate oxidase gene (oxo gene)

[0045] The inventors analyzed the suspected oxo gene sequences that have been functionally verified (such as wheat oxo and barley oxo) and have not been experimentally verified in the NCBI database, and screened to obtain genes that can be used for soluble expression of oxalate oxidase in prokaryotic systems. For known oxo genes and suspected oxo gene sequences in the NCBI database, directly synthesize their CDS sequences, construct E. coli expression vectors, and perform expression tests. For the gene with oxalate oxidase activity, further optimize the codon, the codon fitness index (CAI) in the optimized sequence is higher than that of the original sequence and greater than 0.7, there are no rare codons, and the GC content is between 42-58%. No more than 18bp repeat sequence; these genes can be selected from wheat oxo (GeneBank Accession NO.AJ556991), barley oxo (GeneBank Accession NO.Y14203), worm oxo (C.sub...

Embodiment 2

[0046] Embodiment 2 Design of oxo gene expression cassette

[0047] The invention of the present application has been found through extensive research, in order to promote the soluble expression of Escherichia coli, before and after the artificially synthesized oxalate oxidase gene oxo gene (SEQ ID NO. The protein tag for soluble protein expression is inserted into the E. coli expression vector pET-28a to construct a series of E. coli recombinant expression vectors, and then transformed into the E. coli expression host for expression to obtain soluble oxalate oxidase that can be expressed efficiently without inclusion body; wherein, the expression frame of the oxo gene in the recombinant expression vector has four forms, such as figure 1 shown. The promoter in the oxo gene expression cassette is the T7 promoter in the pET-28a vector.

[0048] Insert the gene tag1 of the protein tag that can promote the expression of the target protein between the oxo gene and the E. coli pro...

Embodiment 3

[0050] Example 3 Construction of oxo gene recombinant expression vector

[0051] The inventors of the present invention conducted extensive studies to find a method for efficiently expressing soluble oxalate oxidase. Specifically, using the oxo gene (Mtoxo) derived from Medicago truncatula as a representative of plant-derived oxalate oxidase, a highly efficient production system was constructed. The types of oxo genes derived from plants and microorganisms are not particularly limited, and any oxo genes identified so far can be used in principle.

[0052] (1) Construction of recombinant expression vector pET28-Mtoxo (oxo expression box 1)

[0053]Using the Mtoxo gene (SEQ ID NO.7) synthesized from the whole gene as a template, design a primer pair F1 / R1 to amplify the Mtoxo gene, and perform gel recovery and purification on the amplified product. The method refers to the commercially available DNA mini-purification reagent According to the method described in the box, the DN...

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Abstract

The invention discloses a recombinant expression cassette for efficiently expressing soluble oxalic acid oxidase and a preparation method of an oxalic acid oxidase capable of efficiently expressing solubility and activity in Escherichia coli. Cellular supernatant is precipitated through ammonium sulfate, and a solution, which is re-dissolved through citric acid-sodium-citrate and contains oxalic acid oxidase, is directly applied to activity determination of oxalate decarboxylase. Compared with traditional methods such as commercially available oxalate determination kits and HPLC (high performance liquid chromatography), the determination method is higher in accuracy and sensitivity, low in time consumption and cost, applicable to high-throughput determination on a large number of samples through a micro-plate reader and high in practicality. The prepared oxalic acid oxidase can be applied to activity determination of enzymes with oxalic acid as substrate or product as well as to determination of the content of the oxalic acid in blood or urine of human or animals as well as various samples containing oxalic acid.

Description

technical field [0001] The invention relates to the field of recombinant protein expression, in particular to a preparation method and application of soluble oxalate oxidase. Background technique [0002] Oxalate oxidase (Oxalate oxidase, OxO, EC1.2.3.4) mainly exists in plants and can decompose oxalic acid into carbon dioxide and hydrogen peroxide. It has great application potential in the diagnosis of diseases related to oxalate accumulation and the clearance of oxalate in the body. [0003] At present, the production of oxalate oxidase mainly includes plant tissue extraction and recombinant expression. Among them, the plant extraction method is greatly affected by raw materials, the content is low, the extraction process is complicated, and the yield is low. Enzymes extracted from plants are generally stably combined with plant cell walls and are solid insoluble substances, which are not convenient for their application in the detection of oxalic acid. Recombinantly exp...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/02C12N9/88C12N1/21C12Q1/527C12Q1/28C12Q1/26C12R1/19
CPCC12N9/0008C12N9/88C12Q1/26C12Q1/28C12Q1/527C12Q2326/96C12Y102/03004C12Y401/01002G01N2333/90203G01N2333/908G01N2333/988
Inventor 汪小锋吴玉峰刘文山汪卫宋保平
Owner WUHAN KANGFUDE BIOTECH CO LTD
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