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Pegylated single-modified recombinant glutathione peroxidase, preparation method and application of pegylated single-modified recombinant glutathione peroxidase

A technology of glutathione peroxidase and glutathione peroxidase, which is used in polyethylene glycol modification of protein compounds, preparation of antioxidant drugs, applications in health care products and cosmetics, preparation, polyethylene glycol The field of alcoholated single-modified recombinant glutathione peroxidase can solve the problems of increased separation and purification challenges and costs, and achieve the effects of prolonging circulation half-life, improving pH tolerance and thermal stability, and resisting protease hydrolysis

Pending Publication Date: 2020-11-06
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, from the attached figure 2 It can be found that this reaction product is actually a mixture of various modified products, which adds challenges and costs to subsequent separation and purification, and the protein modified by this patent is recombinant glutathione peroxidation without cysteine ​​in the molecule GPX1 mutant

Method used

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  • Pegylated single-modified recombinant glutathione peroxidase, preparation method and application of pegylated single-modified recombinant glutathione peroxidase
  • Pegylated single-modified recombinant glutathione peroxidase, preparation method and application of pegylated single-modified recombinant glutathione peroxidase
  • Pegylated single-modified recombinant glutathione peroxidase, preparation method and application of pegylated single-modified recombinant glutathione peroxidase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Example 1: PEGylated Single Modification of Recombinant Glutathione Peroxidase 1

[0085] (1) Acquisition of recombinant glutathione peroxidase 1

[0086] Recombinant glutathione peroxidase 1 (GPX1) was prepared according to the method published in Chinese patent 201510431360.1. GPX1 target proteins were identified by SDS-PAGE and Western blot.

[0087] (2) Modification reaction

[0088] The activated linear monomethoxy polyethylene glycol succinimide succinate with an average molecular weight of 20kD is used as a modifier to covalently couple with the recombinant glutathione peroxidase GPX1 obtained in step (1) . In the reaction, the molar ratio of GPX1 to modifier was 1:30, and the covalent coupling reaction was carried out at 4°C and pH 8.5 for 30 minutes, and linear monomethoxypolyethylene glycol succinimide succinic acid was added The ester molar ratio is 1:1 and the terminator glycine terminates the reaction to obtain a linear monomethoxypolyethylene glycol su...

Embodiment 2

[0092] Example 2: PEGylated Single Modification of Recombinant Glutathione Peroxidase 4

[0093] (1) Acquisition of recombinant glutathione peroxidase 4

[0094] Recombinant glutathione peroxidase 4 (GPX4) was prepared according to the method published in Chinese patent 201510431360.1. First use the gene synthesis method to obtain the hybrid tRNA gene, then synthesize or amplify the GPX4 gene, and assemble the artificially synthesized hybrid tRNA and the gene sequences corresponding to its upstream and downstream promoters and terminators into the polyclonal secretory prokaryotic expression vector site, and then assemble the GPX4 gene into the multiple cloning site of the secretory prokaryotic expression vector, and use the genetic mutation method to mutate the coding sequence of selenocysteine ​​in the active center of GPX4 to TAG, and transform UAG Read through the engineering bacteria, use hybrid tRNA to encode the UAG stop codon as selenocysteine ​​in the nutrient agar me...

Embodiment 3

[0099] Embodiment 3: the physicochemical property of mPEG-GPX

[0100] (1) Circular Dichroism Spectrum

[0101] Circular dichroism analysis was performed using a circular dichroism spectrometer, and the secondary structures of GPX and mPEG-GPX were compared. The GPX samples (GPX1 and mPEG-GPX1, GPX4 and mPEG-GPX4) before and after polyethylene glycol modification were placed in 20mmol / L phosphate buffer at pH 8.5, and measured by BCA method and using 20mmol / L of pH 8.5 L phosphate buffer adjusted the final concentration of each sample to 0.1 mg / mL. Add the sample to a circular dichroic cuvette with a light path of 0.1 cm, place it in a J-810 circular dichroic spectrometer, and set it at room temperature and N 2 measured in the environment. The buffer spectrum served as a blank control and was removed from the spectrum of the protein solution. The wavelength scanning range is 195~250nm. Measure three times and take the average value.

[0102] Depend on Figure 7 It can b...

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Abstract

The invention discloses pegylated single-modified recombinant glutathione peroxidase (mPEG-GPX), and a preparation method and application of the pegylated single-modified recombinant glutathione peroxidase (mPEG-GPX) in preparation of antioxidant drugs, health care products and cosmetics, and belongs to the technical field of biology. The preparation method comprises the following step of carryingout covalent coupling on activated linear-chain monomethoxy polyethylene glycol succinimide succinate (mPEG-SS) and an amino group of recombinant glutathione peroxidase (GPX) to obtain mPEG-GPX. After GPX is modified by polyethylene glycol, the stability of the product mPEG-GPX is obviously enhanced compared with that of the GPX, the in-vivo antioxidant effect of mPEG-GPX is superior to that of the GPX, and development and utilization of mPEG-GPX health care products are facilitated; according to the obtained pegylated single-modified recombinant glutathione peroxidase, the pH tolerance, thethermal stability and the protease hydrolysis resistance of the recombinant glutathione peroxidase are improved, the circulating half-life period of the recombinant glutathione peroxidase in vivo is effectively prolonged, the stability of a drug is improved, and the drug effect is improved.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to polyethylene glycol modified protein compounds, in particular to a class of pegylated monomodified glutathione peroxidase (mPEG-GPX), a preparation method and its use in the preparation of antioxidant Applications in pharmaceuticals, nutraceuticals and cosmetics. Background technique [0002] Glutathione peroxidase is an important class of enzymes, together with superoxide dismutase and catalase, it constitutes the main antioxidant enzyme system in the body. Glutathione peroxidase can block the damage of oxygen free radicals to body tissues by catalyzing the reaction between reduced glutathione and hydrogen peroxide. However, the source of natural glutathione peroxidase is limited and its properties are unstable. To solve this problem, Chinese patent 201510431360.1 discloses a hybrid tRNA and its application in the preparation of glutathione peroxidase. A recombinant glutathione perox...

Claims

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Application Information

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IPC IPC(8): C12N9/08C12N9/96A61K38/44A61K47/60A61K8/66A61P39/06A61Q19/08A23L33/17
CPCC12N9/0065C12N9/96A61K47/60A61K8/66A61P39/06A61Q19/08A23L33/17C12Y111/01009A61K38/00
Inventor 魏景艳王艳伟闫琦
Owner JILIN UNIV
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