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Expressing method of biotinylated ATPS (Aqueous Two-Phase System)

An expression method and biotinylation technology, which is applied in the field of biotinylated ATPS expression, can solve the problems of affecting protein activity, cumbersome operation, and product inhomogeneity, and achieve the effects of reducing synthesis burden, increasing expression, and high yield

Inactive Publication Date: 2016-04-06
BEIJING ZHONGKEZIXIN TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The preparation of biotinylated ATPS by chemical modification has the disadvantages of cumbersome operation and heterogeneous products
In addition, in the process of chemical modification, if the lysine residue in the active center of the protein is modified, it is likely to affect the activity of the protein
Although the biological modification method can avoid the above disadvantages, when the recombinant expression vector constructed by using BCCP87 and ATPS is used for fusion expression, due to the need to express 87 more amino acid residues, the expression level of the fusion protein is reduced to a certain extent; and there are some inclusion bodies Expressed recombinant protein; expressed fusion protein ATPS activity is relatively low
Therefore, it cannot better meet the practical application

Method used

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Effect test

Embodiment 1

[0034] The expression method of embodiment 1 biotinylated ATPS

[0035] Include the following steps:

[0036] ⑴. Introduce NdeI and NotI restriction sites and corresponding protective bases respectively upstream and downstream of the nucleotide sequence shown in SEQ ID NO: 2 to obtain the nucleotide sequence shown in SEQ ID NO: 3, named sequence A1 , chemical total synthesis sequence A1;

[0037] ⑵. Digest the pMAL-p5E plasmid and the sequence A1 obtained in Step 1 of Example 1 with restriction endonucleases NdeI and NotI, and recover the digested product with a DNA gel recovery kit, and digest the nucleotide sequence A1 and The pMAL-p5E plasmid was ligated overnight at 16°C to obtain the recombinant expression vector A1;

[0038] ⑶. Design primer-specific primers, using Saccharomyces cerevisiae genomic DNA as a template, PCR amplification of Saccharomyces cerevisiae ATPS coding gene: the restriction site of the forward primer is EcoRⅤ, the sequence is as shown in SEQIDNO: 4...

Embodiment 2

[0042] The expression method of embodiment 2 biotinylated ATPS

[0043] Include the following steps:

[0044] ⑴. Introduce NdeI and NotI restriction sites and corresponding protective bases respectively upstream and downstream of the nucleotide sequence shown in SEQ ID NO: 2 to obtain the nucleotide sequence shown in SEQ ID NO: 3, named sequence A1 , chemical total synthesis sequence A1;

[0045] ⑵. Digest the pMAL-p5X plasmid and the sequence A1 obtained in Step 1 of Example 2 with restriction endonucleases NdeI and NotI, and recover the digested product with a DNA gel recovery kit, and digest the nucleotide sequence A1 and The pMAL-p5X plasmid was ligated overnight at 16°C to obtain the recombinant expression vector A2;

[0046] ⑶. Design primer-specific primers, use Saccharomyces cerevisiae genomic DNA as a template, and PCR amplify Saccharomyces cerevisiae ATPS coding gene: the restriction site of the forward primer is NotI, and the sequence is shown in SEQ ID NO: 6, and...

Embodiment 3

[0050] Purification of embodiment 3 biotinylated ATPS

[0051] The biotinylated ATPS expressed according to the method of Example 1 or 2 was purified using an Amylose affinity chromatography column.

[0052] Purification method, comprising the following steps:

[0053] ⑴. Centrifuge to collect the cells after induced expression culture, break up the cells by ultrasonic, centrifuge the crushed solution at 4°C and 12000rpm for 30 minutes, and collect the supernatant;

[0054] ⑵. Equilibrate the Amylose affinity chromatography column with 10 times the column bed volume of equilibration buffer (final concentration 15mM Tris-HCl + final concentration 150mMNaCl + final concentration 0.5mMEDTA);

[0055] ⑶. Add the supernatant to the Amylose affinity chromatography column at a flow rate of 1 mL / min to hang the target protein on the column;

[0056] ⑷. Rinse with 20 times column bed volume equilibration buffer until there is basically no impurity protein;

[0057] ⑸. Elute the targ...

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Abstract

The invention provides an expressing method of a biotinylated ATPS (Aqueous Two-Phase System). According to the method, a biotin receptor protein encoding gene (ATG GCT TTC TCT CTG CGF TCT ATC CTG GAA GCT GGT AAA ATG GAA CTG CGT AAC ACC CCG GGT GGT TCT) is constructed into a colibacillus expression vector, so as to obtain a recombinant expression vector; then a brewer yeast ATPS encoding gene is constructed to the downstream of the biotin receptor protein encoding gene in the recombinant expression vector, so as to convert colibacillus host cells and perform inducible expression culture. The biotinylated ATPS expressed according to the method provided by the invention exists in a soluble form, the yield reaches 26.7 to 35.6 mg / 100 mL of culture medium, the specific activity reaches 5.9*104 to 6.8*104 U / mg, and the biotinylated ATPS can be effectively combined with magnetic beads to which avidin is connected and is used for a pyrophosphate sequencing reaction.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to a method for expressing biotinylated ATPS. Background technique [0002] ATPsulfurylase (ATPsulfurylase, ATPS, EC2.7.7.4) is a reversible catalyzed reaction between adenosine-5'-phosphosulfate (APS) and pyrophosphate (pyrophosphate, PPi) to generate adenosine triphosphate (ATP) and A sulfate enzyme that plays an important role in the metabolism of sulfur in organisms. ATP sulfurylase widely exists in plants, animals and microorganisms. At present, researchers have successfully isolated and purified ATP sulfurylase from Saccharomyces cerevisiae, Penicillium chrysogenum, Arabidopsis rhizobium, and terminal thermophilic microorganisms, and successfully cloned ATP sulfuric acid from eukaryotes, plants and animals. Encoding gene of enzyme and realize its heterologous expression. ATP sulfurylase-coupled luciferase can be used in PPi quantification, DNA polymerase enzyme activity measuremen...

Claims

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Application Information

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IPC IPC(8): C12N9/12C12N15/70
CPCC12N9/1241C07K2319/32C12N15/62C12N15/70C12N2800/101C12Y207/07004
Inventor 高静蔡亦梅吴超徐潇张睿王者馥王绪敏殷金龙任鲁风
Owner BEIJING ZHONGKEZIXIN TECH
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