Recombinant buckwheat glutaredoxin as well as preparation method and application thereof

A protein and buckwheat technology, applied in the field of biotechnology and medical and health products, can solve the problems of protein expression, purification and functional analysis that have not been reported yet

Active Publication Date: 2016-11-16
SHANXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Gluredoxin has been found in rice, spinach, Arabidopsis, poplar and other plants, but the identification of buckwheat glutaredoxin (rbGrx) gene, protein expression, purification and functional analysis have not been reported yet.

Method used

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  • Recombinant buckwheat glutaredoxin as well as preparation method and application thereof
  • Recombinant buckwheat glutaredoxin as well as preparation method and application thereof
  • Recombinant buckwheat glutaredoxin as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 Screening and Analysis of Buckwheat Glutenedoxin Gene

[0022] Buckwheat gluten redoxin (rbGrx) gene, the nucleotide sequence of which is SEQ ID NO:1. The gene was screened by analyzing the whole buckwheat gene sequence. Sequence alignment of rbGrx with Grx proteins from different plants (see figure 1 ), which included 12 Grx sequences from nine different plants. Among them, PoGRXC1 is from the National Institutes of Health database 2E7P_A, PoGRXS14 is from the National Institutes of Health database Accession:2LKU_A, PoGRXS12 is from the National Institutes of Health database Accession:3FZ9_A, AtGRXS16 is from the National Institutes of Health database Accession:2LWF_A, AtGRXcp is from the National Institutes of Health database Database Accession: 3IPZ_A, AtGRXC5 from the National Institutes of Health database Accession: 3RHB_A, OrGRX (Oryza sativa) from the National Institutes of Health database Accession: CAA54397, CuGRX (Cucumis sativus) from the National...

Embodiment 2

[0024] Example 2 Expression and purification of recombinant buckwheat glutaredoxin gene in Escherichia coli.

[0025] The rbGrx gene was constructed on the pGEX-6p-1 vector by genetic engineering technology. Transform the fusion protein expression plasmid constructed by cloning into BL21(DE3), inoculate it in 5 mL of LB culture overnight at 37°C, and transfer it to 800 mL of LB medium for expansion after 12 hours, and shake it at 37°C Cultivate in the bottle until the OD is about 0.4-0.6, reduce the culture temperature to 16°C after 4 hours, and then add IPTG (isopropyl-β-D thiogalactopyranoside) with a final concentration of 0.5mM to induce expression, about 16 - Collect bacteria by centrifugation after 20 hours. Use 50mM HEPES buffer (pH 7.8, containing 200mM NaCl) to suspend the cells, use a low-temperature ultra-high pressure cell disruptor to lyse the cells, separate the insoluble precipitate by centrifugation, and pass the supernatant obtained after high-speed centrifug...

Embodiment 3

[0026] Example 3 Determination of rbGrx enzyme activity of recombinant buckwheat gluteledoxin

[0027]Determination of Grx activity of protein: the reaction system is 500 μL, which contains HEPEs (50mmol / L, pH 7.0), EDTA (1mmol / L), GSH (1mmol / L), NADPH (0.25mmol / L), 1U GR (2μmol / L L). Mix the reactants for pre-incubation for 3min, then add a final concentration of 0.5mmol / L HED (bisdihydroxyethyl disulfide) to start the reaction, and monitor the 340nm (ε NADPH =6220M -1 cm -1 ) at the change of NADPH absorbance value. Grx activity is defined as the amount of enzyme required to consume 1 μmol / L NADPH per minute at 25°C, that is, one activity unit. The specific activity of the rbGrx enzyme obtained through measuring is 300U / mg ± 15.0 (see image 3 ).

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Abstract

The invention provides recombinant buckwheat glutaredoxin as well as a preparation method and application thereof. The preparation method comprises the steps of constructing the recombinant buckwheat glutaredoxin (rbGrx) onto a pGEX-6p-1 carrier, transforming into escherichia coli BL21 (DE3), and performing induced expression on fusion protein connected with GST; carrying out enzyme digestion, and then using an anion exchange column and a gel column for further purifying to obtain the rbGrx with the purity of 98% or more. Then, culture is carried out under the crystallization conditions of pH being 4.5, 22% of PEG 3350 (W/V) and 0.1M of CH3COONa, so that a compound crystal of the rbGrx and glutathione (GSH) is obtained; the structure of the compound crystal is measured by an X-ray single-crystal diffraction method, so that important amino acid residues related to catalytic activity can be clearly distinguished, and the stability and enzyme activity are further regulated and controlled purposefully. The rbGrx also has high thioltransferase activity, and the enzyme specific activity is 300U/ mg. The rbGrx can be applied to a catalytic thiol transfer reaction, and the crystal and structure of the rbGrx also have a practical value in the aspects of health care, food development, and the like.

Description

technical field [0001] The invention relates to the fields of biotechnology and medical health products, in particular to glutaredoxin, and more specifically to a recombinant buckwheat glutaredoxin (recombinant buckwheat glutaredoxin, referred to as rbGrx) and its preparation method and application. Background technique [0002] Some proteins in organisms can quickly undergo reversible sulfhydryl and disulfide bond reactions between the active site of cysteine ​​residues and the corresponding disulfide cysteine, thereby regulating the redox of sulfhydryl-containing proteins in cells Potential, the reversible reduction reaction of disulfide bonds is mainly mediated by a series of sulfhydryl oxidoreductases containing disulfhydryl (C-X-X-C) functional regions as active sites. The systems responsible for sulfhydryl redox in organisms mainly include Thioredoxin system and Glutaredoxin system. Grx, glutathione (GSH), glutathione reductase (glutathione reductase, GR) and NAD(P)H ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N15/53C12N9/10C12N15/54C12N15/70C12R1/19
CPCC07K2299/00C12N9/0004C12N9/13C12N15/70C12N2800/101C12Y120/04001
Inventor 王宏飞张新瑀王文明王转花
Owner SHANXI UNIV
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