Directional covalent immobilization method of biotin-protein ligases (BirA) on magnetic microsphere surface

A biotin ligase, magnetic microsphere technology, applied in the field of biochemistry, can solve the problems of no fixed binding mode, affecting the catalytic activity of the enzyme, position uncertainty, etc., to avoid random coupling and improve the use efficiency of enzymes , the effect of high fixed amount

Active Publication Date: 2016-09-21
WEIFANG MEDICAL UNIV
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AI Technical Summary

Problems solved by technology

However, the high-level structure of enzymes is very sensitive to the environment, and physical, chemical and biological factors can cause enzymes to lose their activity.
Chemical-based covalent binding and immobilization behavior by -NH of enzyme protein molecules 2 , -COOH, -OH or -SH and other active groups are covalently bonded to the surface active groups of the solid phase support. Due to the uncertainty and non-uniqueness of the position of the above active groups in the protein molecule, this covalent bond There is no fixed binding mode for valency binding, and the catalytic activity of the enzyme is affected when the linking site is located at the active site of the enzyme or near the group

Method used

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  • Directional covalent immobilization method of biotin-protein ligases (BirA) on magnetic microsphere surface
  • Directional covalent immobilization method of biotin-protein ligases (BirA) on magnetic microsphere surface
  • Directional covalent immobilization method of biotin-protein ligases (BirA) on magnetic microsphere surface

Examples

Experimental program
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Embodiment 1

[0031] Embodiment 1, the construction of recombinant MLAQGS-His-BirA gene prokaryotic expression vector

[0032] According to the BirA gene sequence of Escherichia coli K-12MG1655 (No: EG10123) registered in GenBank, the following primers were designed:

[0033] Primer 1, as shown in SEQ ID No: 1:

[0034] 5'-GAATTCGATGCTAGCGCAGGGATCACACCATCACCATCACCATATGAAGGATAACA-3';

[0035] Primer 2, as shown in SEQ ID No: 2:

[0036] 5′-CCGGGATCCTTTATTTTTCTGCACTACGCAGGG-3′

[0037] Among them, Primer 1 contains EcoR I restriction site, MLAQGS sequence, 6×His sequence, and Primer 2 contains BamH I restriction site.

[0038] Using the E.coli DH5α genome as a template, Primer 1 and Primer 2 were used as primers to amplify the BirA gene by PCR, and the obtained gene fragment was cloned into the pUC18 plasmid EcoR I / BamH I restriction site according to molecular biology methods to obtain pMLAQGS- His-BirA expression plasmid vector.

Embodiment 2

[0039] Embodiment 2, the construction of recombinant BirA-His-MLAQGS gene prokaryotic expression vector

[0040] According to the BirA gene sequence of Escherichia coli K-12MG1655 (No: EG10123) registered in GenBank, the following primers were designed:

[0041] Primer 3, as shown in SEQ ID No: 3:

[0042] 5'-GAATTCGATGAAGGATAACA-3';

[0043] Primer 4, as shown in SEQ ID No: 4:

[0044] 5′-CCGGGATCCCTGGTGCTGGTGCTGGTGTGATCCCTGCGCTAGCATTTATTTTTCTGCACTACGCAGGG-3′

[0045] Among them, Primer 3 contains EcoR I restriction site, and Primer 4 contains 6×His sequence, MLAQGS sequence and BamH I restriction site.

[0046] Using the E.coli DH5α genome as a template, Primer 3 and Primer 4 were used as primers to amplify the BirA gene by PCR, and the obtained gene fragment was cloned into the pUC18 plasmid EcoR I / BamH I restriction site according to molecular biology methods to obtain pBirA- His-MLAQGS expression plasmid vector.

Embodiment 3

[0047] Example 3, Preparation and Purification of Genetic Engineering Technology of Recombinant MLAQGS-His-BirA and BirA-His-MLAQGS

[0048] pMLAQGS-His-BirA plasmid CaCl 2 E.coli BL21(DE3) was transformed by the method to construct pMLAQGS-His-BirA / BL21 engineering bacteria.

[0049] pBirA-His-MLAQGS plasmid CaCl 2 E.coli BL21(DE3) was transformed by the method to construct pBirA-His-MLAQGS / BL21 engineering bacteria.

[0050] The above two kinds of engineered bacteria were activated overnight, transferred to fresh LB medium (ampicillin concentration 100 μg / mL) according to 5% inoculum size, cultured with shaking at 37° C. for 12-16 hours, and centrifuged to collect the bacteria. Resuspend the bacteria in PBS solution containing 20mM imidazole and 500mM NaCl (20mM, pH 8.0), break the wall by ultrasonic, centrifuge at 10000rpm to separate the bacteria fragments, filter the supernatant through a 0.22μm filter membrane, and flow through the HisTrap chromatography column at 0.5m...

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Abstract

The invention discloses a directional covalent immobilization method of biotin-protein ligases (BirA) on a magnetic microsphere surface. The method comprises the following steps: firstly, using a genetic engineering technology to express a biotin-protein ligase recombinant protein of which the N-terminal has an MLAQGS (methionine-leucine-alanine-glutamine-glycine-serine) sequence and a polyhistidine sequence; then utilizing the catalysis of transglutaminase, making -NH2 of glutamine in the biotin-protein ligase recombinant protein and -NH2 on the amino-modified magnetic microsphere surface form covalent connection, thus directionally immobilizing the biotin-protein ligases on the magnetic microsphere carrier surface by a manner of covalent binding. According to the invention, the new method for immobilizing BirA is established, so that the enzymatic catalysis activity of BirA can be fully kept, and can be repeatedly used to improve the use efficiency of BirA and lower the use cost.

Description

technical field [0001] The invention belongs to the field of biochemistry, and in particular relates to a directional immobilized enzyme, that is, biotin ligase (BirA) is directionally fixed on the surface of a magnetic microsphere carrier in a covalently bonded manner by a biological method. Background technique [0002] Biotin ligase, also known as biotin-protein ligase and BirA enzyme, is a bifunctional protein encoded by the BirA gene of Escherichia coli. This enzyme can act as a repressor protein to inhibit the operon of biotin synthesis. More importantly, it can activate biotin to form bioacyl 5'adenylate, and then transfer biotin to biotin receptor protein. By adding a peptide sequence (biotinylated sequence, such as Avi-tag) that can be recognized by BirA enzyme on the target protein, BirA enzyme can bind biotin to the target protein, and the labeling process is an enzymatic reaction. Compared with the most commonly used chemical coupling technology to prepare prote...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/14C12N11/10C12N11/08C07K1/13
CPCC07K1/13C12N9/93C12N11/08C12N11/10C12N11/14C12Y603/04015
Inventor 唐金宝于常梅鲍如梦杨洪鸣
Owner WEIFANG MEDICAL UNIV
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