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Method for detecting OGT enzyme activity in vitro

An in vitro detection and enzyme activity technology, applied in the field of biochemical analysis and detection, can solve the problems of unfriendly environment, the existence of azide synthesis process, and no activity, etc., and achieve the effect of high detection signal intensity, high conversion efficiency and simple operation

Pending Publication Date: 2022-03-04
无锡麦迪科思生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The fluorescence polarization method involves the modification of the donor substrate or the acceptor substrate, which cannot reflect the natural enzyme activity of OGT, and the synthesis of the fluorescent substrate is cumbersome.
In the click chemistry method, a non-natural OGT enzyme donor substrate such as azide-functionalized modified UDP-GlcNAz is used, followed by a click chemical reaction with an alkyne-functional signaling molecule to detect OGT enzyme activity, but this method involves The chemical synthesis steps are cumbersome, and there are safety hazards in the synthesis process of azide, while the production cost is high, and the unnatural donor substrate does not have the same activity as the natural substrate
In addition, it has also been reported to use isotope-labeled donor substrates to detect the activity of OGT on substrate proteins, but it has potential radioactive hazards, is not friendly to the environment, and has high requirements for equipment and environment, so it is difficult to popularize and use.

Method used

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  • Method for detecting OGT enzyme activity in vitro
  • Method for detecting OGT enzyme activity in vitro
  • Method for detecting OGT enzyme activity in vitro

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1 Design and preparation of highly active OGT substrate polypeptide ZO3-S371A

[0069] In this example, the amino acid sequence of the highly active OGT substrate polypeptide ZO3-S371A is derived from the ZO3 protein sequence (NP_001254489.1, SEQ ID No.1) in the NCBI database. Select the amino acid sequence corresponding to positions 357-371 in SEQ ID No.1, change the carboxy-terminal amino acid of the sequence to alanine, and add a glycine and a cysteine ​​to the amino-terminal to obtain the present invention. Highly active OGT substrate polypeptide ZO3-S371A (SEQID No.2) with coupling function. The specific preparation method of ZO3-S371A polypeptide is as follows:

[0070] The preparation of ZO3-S371A polypeptide adopts the principle of polypeptide solid-phase synthesis based on the Fmoc protection strategy, and is synthesized by Symphony polypeptide solid-phase synthesizer. All amino acids constituting the polypeptide of SEQ ID No.2 are standard amino acid...

Embodiment 2

[0075] Example 2 Immobilization and blocking of highly active OGT substrate polypeptide ZO3-S371A

[0076] Dissolve the OGT substrate polypeptide ZO3-S371A in a fixation buffer containing 0.1 mM Na 3 PO 4 , 0.15mM NaCl, 10mM EDTA, pH 7.2, the concentration of the polypeptide substrate after dissolution is 50 ug / ml. The 96-well plate containing activated maleimide groups was purchased from Thermo Fisher Scientific (Cat. No. 15150). Wash the well plate three times with 200ul / well of washing buffer and discard the washing buffer. The wash buffer contains 0.1 mM Na 3 PO 4 , 0.15mM NaCl, 0.05% tween-20, pH 7.2. The polypeptide substrate dissolved in the previous step was incubated with a 96-well plate containing maleimide for 2 hours at a temperature of 25°C. After incubation, the supernatant was discarded, the plate was washed three times with 200ul / well of washing buffer, and the washing buffer was discarded. Configure the blocking solution as 10ug / ml cysteine ​​aqueous sol...

Embodiment 3

[0077] Example 3 Enzymatic reaction of OGT to ZO3-S371A glycosylation

[0078] The buffer system of OGT enzyme reaction is 50mM Tris-HCl, 1mM DTT, 12.5mM MgCl 2 , pH 7.5. The donor substrate for OGT enzyme is UDP-GlcNAc. Add the donor substrate UDP-GlcNAc of OGT to the above-mentioned OGT enzyme reaction system to make its final concentration reach 100um / L, further add purified OGT enzyme 0.5ug or crude enzyme 5ug, and mix well. The above 100ul / well enzyme reaction mixture was added to the 96-well plate immobilized with the OGT substrate polypeptide, the shaking table was rotated at 200rpm, and the temperature was 30°C, and incubated for 3h.

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Abstract

The invention discloses a method for detecting the enzyme activity of O-linked-N-acetylglucosamine transferase OGT in vitro, namely a method for detecting the enzyme activity of O-linked-N-acetylglucosamine transferase OGT in vitro, and the method is low in construction cost, simple to operate and high in detection sensitivity. The method disclosed by the invention comprises the following steps: (1) synthesizing and purifying an OGT high-activity substrate polypeptide ZO3-S371A; (2) carrying out coupling immobilization on the OGT substrate polypeptide ZO3-S371A, and closing a non-specific site of the OGT substrate polypeptide ZO3-S371A; (3) carrying out glycosylation modification on the substrate polypeptide by using the OGT; and (4) detecting the substrate polypeptide subjected to OGT glycosylation through WGA-FITC or WGA-HRP. According to the method, the activity of the substrate polypeptide is high, the preparation cost is low, the detection operation is simple, the sensitivity is high, and the method can be used for measuring the enzyme activity of OGT expressed and purified and OGT in pathological tissue source samples in vitro and can also be used for evaluating the activity of OGT inhibitor drugs.

Description

technical field [0001] The invention belongs to the technical field of biochemical analysis and detection, and in particular relates to a method for detecting OGT enzyme activity in vitro, that is, a method for detecting the enzyme activity of O-linked-N-acetylglucosaminyltransferase OGT. Background technique [0002] O-linked-N-acetylglucosaminyl transferase OGT is responsible for catalyzing the O-linked N-acetyl-glucosamine modification of protein serine and threonine side chain hydroxyls in eukaryotic cells, referred to as O-GlcNAc glycosylation modification. There are many substrate proteins in the human body that can be modified by O-GlcNAc glycosylation mediated by OGT enzymes, and they involve multiple levels of human cell activities and biological reactions, and participate in the regulation of gene expression mediated by important transcription factors such as STAT3 and p53 , regulate the cell cycle mediated by histones and cell cycle proteins, control the biologica...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/573G01N33/543G01N33/535G01N33/533C07K14/705C07K1/04C07K1/06
CPCG01N33/573G01N33/543G01N33/535G01N33/533C07K14/705G01N2333/91102
Inventor 袁脉施杰
Owner 无锡麦迪科思生物科技有限公司
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