Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Application of fba8 gene or fba8 protein in preparation of reagents with phosphotransfer activity and/or proteolysis activity

A technology of proteolysis and protein, applied in the field of multifunctional enzymes

Active Publication Date: 2022-06-24
INST OF FORESTRY CHINESE ACAD OF FORESTRY
View PDF4 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] So far, the reports on the function of FBA8 are limited to its fructose-1,6-bisphosphate aldolase properties, and there are no related reports on other enzymatic properties

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Application of fba8 gene or fba8 protein in preparation of reagents with phosphotransfer activity and/or proteolysis activity
  • Application of fba8 gene or fba8 protein in preparation of reagents with phosphotransfer activity and/or proteolysis activity
  • Application of fba8 gene or fba8 protein in preparation of reagents with phosphotransfer activity and/or proteolysis activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Cloning, Expression and Purification of Multifunctional Enzyme FBA8

[0042] 1. Acquisition of Arabidopsis materials

[0043] Arabidopsis thaliana were cultured in a culture chamber with a temperature of 23°C, a humidity of 50%, and a photoperiod of 16 hr light / 8 hr dark.

[0044] 2. Extraction of rachis stem RNA from Arabidopsis thaliana and reverse transcription into cDNA

[0045] When the plant height is close to 20 cm, cut off the rachis without flowers. After the material was ground with liquid nitrogen, total RNA was extracted with an RNA extraction kit (Adelai Company Plant RNA Rapid Extraction Kit RN38-EASYspinPlus). The extracted total RNA was reversely transcribed into cDNA using a reverse transcription kit (Adler's first-strand reverse transcription kit PC18-TRUEscript 1st Strand cDNASynthesis Kit).

[0046] 3. Target gene cloning

[0047] (1) Primer design

[0048] FBA8 (AT3G52930) gene sequence encodes 358 amino acids, and its CDS and amino acid sequen...

Embodiment 2

[0069] Heterologously expressed FBA8 has fructose-1,6-bisphosphate aldolase activity and its influencing factors.

[0070] The FBA8 protein solution obtained from Example 1 was used to detect the aldolase activity with an aldolase detection kit (BC2000 from Soleibo Company). The final concentration of FBA8 was 0.18 mg / mL, and the incubation time was 1 hour. figure 2 It was shown that FBA8 with aldolase activity can be obtained by the above heterologous expression system.

[0071] Application of aldolase detection kit to detect ATP, EDTA, CaCl 2 , The effect of BIN2 protein (an interacting protein of FBA8, AT4G18710) on the activity of FBA8 aldolase. The final concentration of FBA8 is 0.18 mg / mL, the final concentration of ATP is 55.6 μM, the final concentration of EDTA is 27.8 mM, and the final concentration of CaCl 2 The final concentration of BIN2 is 0.56 mM and the final concentration of BIN2 is 0.06 mg / mL. like figure 2 As shown, EDTA can nearly double the activity o...

Embodiment 3

[0073] Heterologously expressed FBA8 has kinase activity and influencing factors.

[0074] The FBA8 protein solution (final concentration 0.24 mg / mL) obtained from Example 1 was added to the kinase reaction system (25 mM Tris-HCl, pH 7.5, 0.5 mM DTT, 10 mM MgCl 2 , 1mMATP), incubated at 37°C for 1 hour, and then commissioned Beijing Qinglian Biotech Co., Ltd. to carry out LC-MS / MS mass spectrometry detection, and it was detected that the FBA8 protein itself has many phosphorylation sites. like image 3 Shown (pep peptide containing this site is not phosphorylated; pep-Pi peptide containing this site is phosphorylated at this site; 2pep means mass spectrometry detected 2 peptides containing this site but not phosphorylated , other numbers +pep and so on; 1pep-Pi means that mass spectrometry detects 1 polypeptide containing phosphorylated at this site, other numbers +pep-Pi and so on; 2pep+1pep-Pi means mass spectrometry detects 2 peptides at the same time A polypeptide that c...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
concentrationaaaaaaaaaa
Login to View More

Abstract

The invention relates to the application of FBA8 gene or FBA8 protein in the preparation of reagents with phosphotransfer activity and / or proteolysis activity, and belongs to the technical field of multifunctional enzymes. The FBA8 gene or FBA8 protein can be used to prepare reagents with phosphotransfer activity and / or proteolytic activity, and the present invention can significantly improve the aldolase activity of FBA8; can maximize the kinase activity of FBA8, and can also close all its activities; The protease activity of FBA8 can be maximized.

Description

technical field [0001] The present invention relates to the technical field of multifunctional enzymes, in particular to the application of FBA8 gene or FBA8 protein in the preparation of reagents with aldolase activity, phosphorylation transfer activity and / or proteolytic activity. Background technique [0002] FBA8 (AT3G52930) is a fructose-1,6-bisphosphate aldolase in Arabidopsis thaliana, which is a key enzyme in the process of glycolysis. FBA8 can decompose fructose-1,6-bisphosphate into dihydroxyacetone phosphate and glyceraldehyde-3-phosphate, and glyceraldehyde-3-phosphate can be further oxidized to 1,3-bisphosphoglycerate and generate NADH. 1,3-Diphosphoglycerate can in turn transfer high-energy phosphate bonds to ADP through substrate-level phosphorylation, thereby generating glycerate-3-phosphate and ATP. [0003] In 1924, the German physiologist Warburg discovered that cancer cells consume a large amount of glucose through the glycolysis pathway compared to othe...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/88C12N9/12C12N9/50
CPCC12N9/88C12N9/12C12N9/63C12Y401/02013
Inventor 国鹏张建国
Owner INST OF FORESTRY CHINESE ACAD OF FORESTRY
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com