Polyethylene glycol-modified recombinant glutathione peroxidase GPx1 mutant, preparation and antioxidant application methods thereof

A technology of glutathione peroxide and polyethylene glycol, applied in biochemical equipment and methods, oxidoreductase, medical preparations of non-active ingredients, etc., can solve the problem of reduced enzyme activity, short half-life, and stability Low-level problems, to achieve the effect of eliminating the antibody reaction of enzyme protein antigen

Pending Publication Date: 2018-11-27
JILIN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the sulfhydryl group of the enzyme is easily oxidized at room temperature, resulting in r

Method used

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  • Polyethylene glycol-modified recombinant glutathione peroxidase GPx1 mutant, preparation and antioxidant application methods thereof
  • Polyethylene glycol-modified recombinant glutathione peroxidase GPx1 mutant, preparation and antioxidant application methods thereof
  • Polyethylene glycol-modified recombinant glutathione peroxidase GPx1 mutant, preparation and antioxidant application methods thereof

Examples

Experimental program
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Example Embodiment

[0062] Example 1 Synthesis of activated linear monomethoxy polyethylene glycol succinimide butyrate.

[0063] 1. Obtained by the following methods:

[0064] (1) Weigh 2.0 g of monomethoxy polyethylene glycol (mPEG) with a molecular weight of 5000, add 4 mL of N,N-dimethylformamide to the flask, and fully dissolve it.

[0065] (2) Add 0.12g of succinic anhydride, mix well, and seal.

[0066] (3) The mixture was reacted at 100°C for 3 hours.

[0067] (4) After cooling the above reaction solution to room temperature, add 0.466g of N-hydroxysuccinimide, 0.825g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and heat to It dissolves.

[0068] (5) React overnight at 30°C for 12 hours, then cool to room temperature.

[0069] (6) Add 40 mL of ether dropwise to the cooled reaction solution at 4°C, filter the resulting precipitate, and dry in vacuum. The dried product was dissolved in dichloromethane and then precipitated with ether. Repeat this process 4 times.

[0070] (7) The res...

Example Embodiment

[0078] Example 2

[0079] Performance evaluation of modified products of mPEGylated recombinant glutathione peroxidase GPx1 mutant:

[0080] (1) When the mass ratio of recombinant glutathione peroxidase GPx1 mutant to linear monomethoxy polyethylene glycol succinimidyl butyrate is 1:60, the thermal stability of the obtained enzyme is improved.

[0081] The reaction system is 500μL, which contains 50mmol / L pH 7.4 PBS, 1mmol / L EDTA, 1mmol / LGSH, 0.25mmol / L NADPH, 1U glutathione reductase and 20-50nM protein sample. The reaction mixture is pre-heated at 19~55℃ for 5min, and then the final concentration is 0.5mmol / L H 2 O 2 Start the reaction. Monitor the change of NADPH absorbance value at 340nm by UV spectrophotometer. Enzyme activity is defined as the amount of enzyme required for the protein to oxidize 1 μmol NADPH per minute at 37°C, and it is expressed as 100% enzyme activity. Such as Figure 5 As shown, at 55°C, the recombinant glutathione peroxidase GPx1 mutant maintained a rel...

Example Embodiment

[0083] Example 3

[0084] The modified product of mPEGylated recombinant glutathione peroxidase GPx1 mutant (mPEG-GPx1) freeze-dried protective agent screening:

[0085] In a 4 degree chromatography refrigerator, GPx1 and mPEG-GPx1 were dialyzed in a solution (200 mL) containing 10% mannitol and 5% glycine (W / V) freeze-dried protective agent for 4 hours, and the dialysate was changed 4 times in the middle. After the dialysis is completed, pre-freeze at -20°C for 12 hours and freeze-dry in a Zirbus Voco 5 freeze dryer; GPx1 and mPEG-GPx1 samples without lyophilization protection agent are pre-freeze at -20°C for 12 hours, It was freeze-dried in a Zirbus Voco 5 freeze-drying machine; according to the enzyme activity determination method in Example 2, the enzyme activity at 37°C was measured. The enzyme activity of the sample before freeze-drying is calculated as 100%.

[0086] After lyophilizing GPx1 and mPEG-GPx1 directly, the enzyme activities maintained 75.36% and 82.42% respectiv...

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Abstract

A polyethylene glycol-modified recombinant glutathione peroxidase GPx1 mutant, a preparation method thereof, and antioxidant application of a recombinant glutathione peroxidase GPx1 mutant unmodifiedand modified by polyethylene glycol belongs to the field of biological technology. Activated linear monomethyl polyethylene glycol succinimide butyrate serves as a modifier and reacts with the recombinant glutathione peroxidase GPx1 mutant at 4-37 DEG C and pH of 6.0-9.0 for 10-60 min, then glycine as a terminating agent is added to terminate the reaction, side products are removed via dialysis toobtain the polyethylene glycol-modified recombinant glutathione peroxidase GPx1 mutant; the applied weight ratio of the recombinant glutathione peroxidase GPx1 mutant and the linear monomethyl polyethylene glycol succinimide butyrate is 1:10-120. Measurement shows that the amino-group modification rate can reach up to 98.73%, and when the amino-group modification rate is higher than 40%, the polyethylene glycol-modified linear monomethyl polyethylene glycol succinimide butyrate can completely eliminate antigen-antibody reaction of foreign proteins, thereby having the advantage of eliminatingthe antigen-anti-body reaction of zymoproteins.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a polyethylene glycol-modified recombinant glutathione peroxidase GPx1 mutant, a preparation method and polyethylene glycol for the recombinant glutathione peroxidase GPx1 mutant Antioxidant application before and after modification. Background technique [0002] Glutathione is the substrate of the selenium-containing glutathione peroxidase GPx1, and selenocysteine ​​SeCys is its catalytic group. GPx, superoxide dismutase SOD, and H2O2 enzyme CAT are three important antioxidant enzymes existing in biological tissues. GPx1 is one of the important subtypes of selenium-containing GPx. It plays an important role in the antioxidant system. It can remove H2O2 and various hydroperoxides in the body, block active oxygen ROS, prevent lipid peroxidation, and treat diseases caused by active oxygen. Aging, cataracts, tumors and cardiovascular and cerebrovascular diseases. GPx1 can not only sc...

Claims

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Application Information

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IPC IPC(8): C12N9/08C12N9/96A61K38/44A61K47/60A61P39/06
CPCA61K47/60A61P39/06C12N9/0065C12N9/96C12Y111/01009A61K38/00
Inventor 魏景艳张广远宋健
Owner JILIN UNIV
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