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A Molecular Probe for Higher Plant Cobalt-Independent Methionine Synthetase and Its Application

A technology of methionine synthase and higher plants, applied in the field of highly selective probes, can solve problems such as unclear binding of probes, and achieve the effect of high specificity and high affinity

Active Publication Date: 2022-03-25
CHANGSHU INSTITUTE OF TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] CN 105503879 A discloses that the structure of the probe G1-yne is similar to the compound of the present application, and it has been reported to covalently modify the tyrosine 111 residue of the Schistosoma japonicum sjGST protein, so it can be used for labeling and immobilization of GST fusion proteins , but the combination of the probe in the eukaryotic proteome including higher plants is not clear (Bioconjugate Chem.2014,25,1911-1915)

Method used

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  • A Molecular Probe for Higher Plant Cobalt-Independent Methionine Synthetase and Its Application
  • A Molecular Probe for Higher Plant Cobalt-Independent Methionine Synthetase and Its Application
  • A Molecular Probe for Higher Plant Cobalt-Independent Methionine Synthetase and Its Application

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Experimental program
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Embodiment

[0046] Biological experiments

[0047] Protein labeling of Arabidopsis thaliana tissue crude extracts based on fluorescent scanning of gel electrophoresis.

[0048] in reaction buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM MgCl 2), the Arabidopsis tissue crude protein was diluted to a final concentration of 2.5 μg / μL and the probe G1-yne was added at a final concentration of 1 μM for 1 hour at room temperature. 1% final SDS was then added to the protein sample and a "click" reaction was performed: for each reaction, 0.2 μL of TAMRA-N was added to 19.2 μL of the protein sample 3 (10mM stock in DMSO, Lumiprobe), CuSO 4 (100 mM stock in water), THPTA (10 mM stock in water, Sigma) and sodium ascorbate (100 mM stock in water). The samples were incubated at room temperature for 1 hour in the dark, 5 μL of 5×SDS loading buffer was added and the reaction was stopped by boiling at 95°C for 15 minutes. Samples were applied to 10% Bis-tris denaturing gels and in-gel fluorescence sca...

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Abstract

The invention belongs to the field of biotechnology, and relates to a highly selective probe for cobalt-independent methionine synthase in higher plants and its application. The present invention provides a highly selective probe for higher plant non-cobalt-dependent methionine synthetase, which has a sulfonyl fluoride group and can specifically and covalently detoxify at lysine residues. tagged protein. The invention can be used to detect the activity, expression level and tissue and subcellular localization of cobalt-independent methionine synthetase in higher plants. The probe of the present invention provides a tool for targeted protein research, enabling deeper research on protein expression changes and protein network maps of different plant types, different strains or different developmental stages.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a highly selective probe of cobalt-independent methionine synthase in higher plants and its application. Background technique [0002] Cobalamin-independent methionine synthase (MS) is widely present in higher plants, and it catalyzes the transfer of methyl groups from N5-methyltetrahydrofolate to homocysteine ​​to generate methionine and tetrahydrofolate. It directly participates in methionine cycle, folic acid cycle and sulfur-containing amino acid metabolism, and is closely related to DNA and protein synthesis and biological methylation, and provides intermediate products for methylation reaction in vivo and the synthesis of polyamine and ethylene. [0003] At present, there are no targeted antibodies and molecular probes specifically for cobalt-independent methionine synthase on the market. Therefore, there is a lack of effective antibody tools for this research field, which can be...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C09K11/06G01N21/64
CPCC09K11/06G01N21/6428G01N21/6458G01N2021/6439C09K2211/1074
Inventor 周怡青陈鑫许静远王春夏戴含章沈陈金鑫
Owner CHANGSHU INSTITUTE OF TECHNOLOGY
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