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A kind of vitamin k epoxide reductase in vitro activity assay method and its application

An epoxide and activity measurement technology, applied in the field of biochemistry, can solve the problems of complicated operation, inability to detect real-time activity, inconvenient high-throughput measurement, etc., and achieve the effects of simple method steps, strong operability, and small reaction system.

Active Publication Date: 2022-07-19
HENAN UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the traditional method is to measure the activity of VKOR protein by extracting microsomes. This method is cumbersome and inconvenient for high-throughput measurement. It can only detect the activity of catalytic KO, but cannot detect the activity of catalytic K, and the activity cannot be detected in real time (Fasco , M.J., Principe, L.M., Walsh, W.a & Friedman, P.a. Warfarin inhibition of vitamin K 2,3-epoxide reductase in rat liver microsomes. Biochemistry. 22, 5655–60 (1983); Rost, S., Fregin, A., Ivaskevicius, V., Conzelmann, E. & Hortnagel, K. Mutations in VKORC1 cause warfare resistance and multiple coagulationfactor deficiency type 2. Nature. 427, 537–541 (2004).)

Method used

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  • A kind of vitamin k epoxide reductase in vitro activity assay method and its application
  • A kind of vitamin k epoxide reductase in vitro activity assay method and its application
  • A kind of vitamin k epoxide reductase in vitro activity assay method and its application

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preparation example Construction

[0066] The preparation method of the purified human VKOR protein used in the examples and test examples is as follows: the gene encoding the human VKOR protein (Gene ID: 79001) is cloned into the Pichia pastoris expression vector pPICZ-B (invitrogen), and its C-termini are connected in sequence PreScission protease cleavage site and GFP-HIS10 tag; protein expression and purification were performed according to the kit (Cat. No. K171001, Thermofisher) and literature (Ren F, Logeman BL, Zhang X, Liu Y, Thiele DJ, YuanP. X-ray structures of The high-affinity copper transporter Ctr1.NatCommun.2019,27;10(1):1386.doi:10.1038 / s41467-019-09376-7.) was performed in the method for expressing membrane proteins in Pichia pastoris. The purification steps are as follows:

[0067] 1) Yeast cells expressing human VKOR protein were crushed and dissolved in lysis buffer (2% DDM, 150 mM NaCl, 50 mM Tris-HCl pH 8.0, 10 μg / mL DNase I, 2 mM PMSF protease inhibitor, the solvent was water), Stir and...

Embodiment 1

[0072] In the present embodiment, the in vitro activity assay method of vitamin K epoxide reductase comprises the following steps:

[0073] 1) Preparation of materials:

[0074] Prepare buffer 1, buffer 2, buffer 3 and buffer 4 respectively, and store them for later use;

[0075] The composition of the buffer solution 1 is: Tris-HCl 20mmol / L of pH 7.5, NaCl 100mmol / L, LMNG 1g / L, and the solvent is water;

[0076] The composition of the buffer solution 2 is: GSH 0.5mol / L, appropriate amount of NaOH, and the solvent is water; pH value is 7.5;

[0077] The composition of the buffer solution 3 is: vitamin K epoxide 1mmol / L, and the solvent is isopropanol;

[0078] The composition of the buffer solution 4 is: vitamin K 1 mmol / L, and the solvent is isopropanol;

[0079] 2) Preparation of working solution:

[0080] Prepare vitamin K epoxide working solution, vitamin K working solution and VKOR protein working solution respectively, and reserve;

[0081] The preparation method of t...

Embodiment 2

[0094] In the present embodiment, the in vitro activity assay method of vitamin K epoxide reductase comprises the following steps:

[0095] 1) Preparation of materials:

[0096] Prepare buffer 1, buffer 2, buffer 3 and buffer 4 respectively, and store them for later use;

[0097] The composition of the buffer solution 1 is: pH 7.0 Tris-HCl 30mmol / L, NaCl 120mmol / L, LMNG 0.5g / L, and the solvent is water;

[0098] The composition of the buffer solution 2 is: GSH 0.4mol / L, appropriate amount of NaOH, and water as the solvent; pH value 7.0;

[0099] The composition of the buffer solution 3 includes: vitamin K epoxide 20mmol / L, and the solvent is isopropanol;

[0100] The composition of the buffer solution 4 includes: vitamin K 50mmol / L, and the solvent is isopropanol;

[0101] 2) Preparation of working solution:

[0102] Take buffer 1, buffer 2 and buffer 3, respectively, to prepare a vitamin K epoxide working solution with a GSH concentration of 60 mmol / L and a vitamin K epox...

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Abstract

The invention discloses a method for measuring the in vitro activity of vitamin K epoxide reductase and its application, and belongs to the technical field of biochemistry. In the vitamin K cycle, γ-glutamate carboxylase (GGCX) utilizes dihydrovitamin K (KH 2 ) is a cofactor to catalyze the γ-carboxylation of the glutamic acid residue in the Gla domain of coagulation factor, while the KH 2 Converted to vitamin K epoxide (KO); vitamin K epoxide reductase (VKOR) catalyzes KO to K, and K to KH 2 , so that vitamin K can be recycled. The detection principle of the present invention is to use the product KH 2 Fluorescence can be generated under the excitation light of 250nm, by detecting the product KH 2 Compared with the traditional microsomal method, it can detect the activity of VKOR protein catalyzing KO or K in a rapid, real-time and high-throughput manner; using this method can effectively Screening small molecule inhibitors targeting VKOR.

Description

technical field [0001] The invention relates to a method for measuring the in vitro activity of vitamin K epoxide reductase and its application, and belongs to the technical field of biochemistry. Background technique [0002] Vitamin K epoxide reductase (VKOR) is a specific target of clinical anticoagulant drug warfarin. Warfarin inhibits the conversion of KO to K and KH by inhibiting the activity of VKOR 2 , thereby blocking the circulation of vitamin K, and then inhibiting the γ-carboxylation modification of the glutamic acid residue in the Gla domain of the coagulation factor, so as to achieve the purpose of anticoagulation. The method of measuring VKOR protein activity in vitro is an important means to study the properties and inhibitors of VKOR protein, and it can also be used to screen novel small molecule inhibitors targeting VKOR. At present, the traditional method is to measure the activity of VKOR protein by extracting microsomes, which is cumbersome and inconve...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/26
CPCC12Q1/26
Inventor 沈国民高蒙刘红丽沈滟曹青李三强
Owner HENAN UNIV OF SCI & TECH
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