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Expression method for heparin precursor sulphation modification enzyme

A technology of heparin precursor and expression method, applied in biochemical equipment and methods, enzymes, enzymes and other directions, can solve problems such as difficult chemical synthesis, and achieve the effects of low cost and simple method

Inactive Publication Date: 2017-05-31
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, for the synthesis of heparin with a molecular weight above pentasaccharide, it is very difficult to use chemical synthesis alone

Method used

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  • Expression method for heparin precursor sulphation modification enzyme
  • Expression method for heparin precursor sulphation modification enzyme
  • Expression method for heparin precursor sulphation modification enzyme

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] (1) Construction of recombinant plasmid: Artificially synthesized 6-ost-3 sequence with NdeI and XhoI restriction sites at both ends, inserted into the NdeI / XhoI site of expression plasmid pET-28a after double digestion, and obtained expression Plasmid pET-28a / 6-ost-3.

[0020] (2) Construction of engineering bacteria: the expression plasmid pET-28a / 6-ost-3 was transformed into Escherichia coli BL21 competent, and the recombinant engineering bacteria were obtained by screening on a kanamycin resistance plate.

[0021] (3) Induced expression of engineered bacteria: Inoculate engineered bacteria into LB medium with shaking at 37°C overnight as seed liquid, take 1 mL of seed liquid and inoculate into 100 mL of LB fermentation medium with shaken culture at 37°C, when the absorbance of the bacteria liquid reaches At 0.6, IPTG was added to induce at 20°C for 10 hours, and the cells were collected by centrifugation at 8000 rpm for 10 minutes.

[0022] (4) Bacterial cell disru...

Embodiment 2

[0025] (1) Construction of recombinant plasmid: Artificially synthesized 6-ost-3 sequence with NdeI and XhoI restriction sites at both ends, inserted into the NdeI / XhoI site of expression plasmid pET-28a after double digestion, and obtained expression Plasmid pET-28a / 6-ost-3.

[0026] (2) Construction of engineering bacteria: the expression plasmid pET-28a / 6-ost-3 was transformed into Escherichia coli BL21 competent, and the recombinant engineering bacteria were obtained by screening on a kanamycin resistance plate.

[0027] (3) Induced expression of engineered bacteria: Inoculate engineered bacteria into LB medium with shaking at 37°C overnight as seed liquid, take 1 mL of seed liquid and inoculate into 100 mL of LB fermentation medium with shaken culture at 37°C, when the absorbance of the bacteria liquid reaches At 0.4, add IPTG with a final concentration of 0.8mmol / L to induce at 20°C for 12h, and centrifuge at 8000rpm for 10min to collect the bacteria.

[0028] (4) Bacte...

Embodiment 3

[0031] (1) Construction of recombinant plasmid: Artificially synthesized 6-ost-3 sequence with NdeI and XhoI restriction sites at both ends, inserted into the NdeI / XhoI site of expression plasmid pET-28a after double digestion, and obtained expression Plasmid pET-28a / 6-ost-3.

[0032] (2) Construction of engineering bacteria: the expression plasmid pET-28a / 6-ost-3 was transformed into Escherichia coli BL21 competent, and the recombinant engineering bacteria were obtained by screening on a kanamycin resistance plate.

[0033] (3) Induced expression of engineered bacteria: Inoculate engineered bacteria into LB medium with shaking at 37°C overnight as seed liquid, take 1 mL of seed liquid and inoculate into 100 mL of LB fermentation medium with shaken culture at 37°C, when the absorbance of the bacteria liquid reaches At 0.6, IPTG with a final concentration of 1.0 mmol / L was added to induce at 20°C for 12 hours, and the cells were collected by centrifugation at 8000 rpm for 10 mi...

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Abstract

The invention relates to an expression method for a heparin precursor sulphation modification enzyme, and relates to the technical field of biotechnology, particularly to a new nucleotide sequence, construction of a pronucleus expression system and a preparation method. An artificially synthesized 6-ost-3 sequence is connected to a pET-28a expression carrier, is converted into an Escherichia coli BL21 competent cell and expresses intracellular soluble protein through the induction of IPTG (Isopropyl Thiogalactoside). The cell is broken by lysozyme; supernatant is collected; the supernatant is purified through a nickel column to obtain 6-OST-3 apoenzyme; the specific enzyme activity of the 6-OST-3 apoenzyme is 1.53 U / mg. According to the expression method for the heparin precursor sulphation modification enzyme, the 6-OST-3 apoenzyme is expressed and prepared by using Escherichia coli; the expression method has the advantages of simple experimental conditions and low cost; the 6-OST-3 serves as a tool enzyme and has an important significance for modification and synthesis of macromolecular heparin.

Description

technical field [0001] The invention discloses a method for expressing a heparin precursor sulfate modification enzyme, which relates to the field of biotechnology, and specifically relates to a new nucleotide sequence and a prokaryotic expression system construction and preparation method. Background technique [0002] Heparan sulfate (HS) is a polysulfated polysaccharide that widely exists on the cell surface and extracellular matrix, and participates in a variety of biological reactions, such as embryonic development, blood coagulation, and viral infection. Studies have found that heparan sulfate has a variety of biological activities, including anticoagulant, antitumor, antiviral, etc., and its biological functions are closely related to the modified sulfate groups on polysaccharides. [0003] Currently, heparin oligosaccharides are mainly prepared by chemical synthesis. However, it is very difficult to synthesize heparin with a molecular weight of more than five sugars...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/70
CPCC12N9/13C12N15/70C12Y208/02C12N2800/101
Inventor 尹鸿萍杨轶伟王莹黄凤杰
Owner CHINA PHARM UNIV