A kind of expression method of biotinylated inorganic pyrophosphatase

A technology of inorganic pyrophosphatase and expression method, which is applied in the field of expression of biotinylated inorganic pyrophosphatase, and can solve the problems of affecting the catalytic activity of enzyme protein, reducing enzyme activity, loss and the like

Active Publication Date: 2017-05-31
BEIJING ZHONGKEZIXIN TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As a biologically active protein, an enzyme is very sensitive to factors such as organic reagents. However, it is often necessary to introduce a large amount of organic reagents during the chemical modification process, which may reduce or even lose the enzyme activity; during the chemical modification process, if the protein is in The modification of the lysine residue in the active center may also affect the catalytic activity of the enzyme protein; in addition, the chemical modification method also has the disadvantages of cumbersome operation and heterogeneous products

Method used

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  • A kind of expression method of biotinylated inorganic pyrophosphatase
  • A kind of expression method of biotinylated inorganic pyrophosphatase
  • A kind of expression method of biotinylated inorganic pyrophosphatase

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Embodiment 1

[0038] The expression method of embodiment 1 biotinylated inorganic pyrophosphatase

[0039] Include the following steps:

[0040] ⑴. Introducing NcoI and HindIII 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 the sequence BAP23-1, chemically synthesized sequence BAP23-1;

[0041] (2) Digest the pET30a(+) plasmid and the sequence BAP23-1 obtained in step (1) of Example 1 with restriction endonucleases NcoI and HindIII, and recover the digested product with a DNA gel recovery kit, and digest the nucleotides Sequence BAP23-1 and pET30a(+) plasmids were ligated overnight at 16°C to obtain recombinant expression vector A1;

[0042] ⑶. Design a pair of primer-specific primers, use Thermococcus litoralis genomic DNA as a template, PCR amplify the coding gene of inorganic pyrophosphatase (GenBank number WP_004067338) and recover ...

Embodiment 2

[0046] Embodiment 2 Purification of biotinylated inorganic pyrophosphatase

[0047] The biotinylated inorganic pyrophosphatase expressed according to the method of Example 1 was purified by using a histidine affinity chromatography column.

[0048] The purification method comprises the following steps:

[0049] ⑴. Centrifuge to collect the bacterial cells after the induction culture in Step ⑹ of Example 1, crush the bacterial cells with ultrasonic waves, centrifuge the crushed liquid at 4°C and 12000rpm / min for 30min, and collect the supernatant;

[0050] ⑵. Equilibrate the histidine affinity chromatography column with 5 times the volume of the column bed (50mM pH8.0 sodium phosphate buffer + 300mM sodium chloride + 2mM imidazole);

[0051] (3) Add the supernatant obtained in step (1) of Example 2 to a histidine affinity chromatography column at a flow rate of 1 mL / min, so that the biotinylated inorganic pyrophosphatase is attached to the column;

[0052] ⑷. Rinse the chroma...

Embodiment 3

[0055] Embodiment 3 Expression method of biotinylated inorganic pyrophosphatase

[0056] Include the following steps:

[0057] ⑴. Introducing the restriction sites of BamHI and XhoI and the 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: 6, Named as sequence BAP23-2, chemically synthesized sequence BAP23-2;

[0058] (2) Digest the pET22b(+) plasmid and the sequence BAP23-2 obtained in Step 1 of Example 3 with restriction endonucleases BamHI and XhoI, and recover the digested product with a DNA gel recovery kit, and digest the nucleotides Sequence BAP23-2 and pET22b(+) plasmids were ligated overnight at 16°C to obtain recombinant expression vector A2;

[0059] ⑶. Design a pair of primer-specific primers, use Saccharomyces cerevisiae genomic DNA as a template, PCR amplify wherein the coding gene of inorganic pyrophosphatase (GenBank numbering is NP_009565)...

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Abstract

The invention belongs to the biotechnology field, and particularly relates to a biotinylation inorganic pyrophosphatase expression method. According to the method, a biotin receptor protein BAP23 composed of 23 amino acid residues is used, the encoding gene of BAP23 is built in an escherichia coli expression vector to obtain a recombinant expression vector, and after an inorganic pyrophosphatase encoding gene is inserted into the recombinant expression vector, an expressed fusion protein can be subjected to biotinylation modification in escherichia coli cells. The yield of biotinylation inorganic pyrophosphatase expressed with the method reaches 21-38 mg / 100 mL medium, and specific activity reaches 94.2-151.6 U / mg. After Bead-LUC obtained after expressed biotinylation inorganic pyrophosphatase is fixed through magnetic beads is washed three times, catalytic activity is not affected basically, and relative activity which is over 91% of activity before washing can still be maintained after 15 times of washing.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for expressing biotinylated inorganic pyrophosphatase. Background technique [0002] Inorganic pyrophosphatase (Inorganic Pyrophosphatase, PPase, EC3.6.1.1) is a class of enzymes that can catalyze the hydrolysis of pyrophosphate to orthophosphate. Pyrophosphoric acid (PPi) is a by-product of DNA and RNA polymerization, amino acid activation, aminoacyl tRNA synthesis, fatty acid β-oxidation, fatty acyl-CoA synthesis, cellulose synthesis, glucose synthesis, and starch synthesis in vivo. , PPase can decompose PPi to make the above reaction to the direction of synthesis and provide energy for various biosynthetic reactions. PPase exists widely in nature and is found in animals, plants and microorganisms. With the development of technical means, PPase has been successfully isolated from Japanese blood sucker, rice, barley, tobacco, yeast, Bacillus subtilis, Escherich...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/14C12N15/70
CPCC12N9/14C12Y306/01001
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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