Application of FBA8 gene or FBA8 protein in preparation of reagent with phosphate transfer activity and/or proteolytic activity

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

Active Publication Date: 2021-10-22
INST OF FORESTRY CHINESE ACAD OF FORESTRY
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  • 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

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  • Application of FBA8 gene or FBA8 protein in preparation of reagent with phosphate transfer activity and/or proteolytic activity
  • Application of FBA8 gene or FBA8 protein in preparation of reagent with phosphate transfer activity and/or proteolytic activity
  • Application of FBA8 gene or FBA8 protein in preparation of reagent with phosphate transfer activity and/or proteolytic 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 was cultured in a culture room with a temperature of 23° C., a humidity of 50%, and a photoperiod of 16 hours light / 8 hours dark.

[0044] 2. Extraction of Arabidopsis inflorescence axis stem RNA and reverse transcription into cDNA

[0045] When the plant height is close to 20 cm, the inflorescence axis stems without flowers are intercepted. After the material was crushed with liquid nitrogen, the total RNA was extracted with an RNA extraction kit (Adelaide Plant RNA Rapid Extraction Kit RN38-EASYspinPlus). The extracted total RNA was reverse-transcribed into cDNA using a reverse transcription kit (PC18-TRUEscript 1st Strand cDNA Synthesis Kit from Adelaide).

[0046] 3. Target gene cloning

[0047] (1) Primer design

[0048] The FBA8 (AT3G52930) gene sequence encodes 358 amino acids, and its CDS and amino acid sequences are show...

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 tested for aldolase activity with an aldolase detection kit (BC2000 from Solebol Corporation), the final concentration of FBA8 was 0.18 mg / mL, and the incubation time was 1 hour. figure 2 It shows that FBA8 with aldolase activity can be obtained through the above-mentioned 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 0.18mg / mL, the final concentration of ATP is 55.6μM, the final concentration of EDTA is 27.8mM, CaCl 2 The final concentration of BIN2 is 0.56mM, and the final concentration of BIN2 is 0.06mg / mL. Such as figure 2 As shown, EDTA can nearly double the activity of FBA8; ATP, CaCl 2 and ...

Embodiment 3

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

[0074] Add the FBA8 protein solution (final concentration 0.24mg / mL) obtained from Example 1 into the kinase reaction system (25mM Tris-HCl, pH 7.5, 0.5mM DTT, 10mM MgCl 2 , 1mMATP), incubated at 37°C for 1 hour, and then entrusted Beijing Qinglian Biotechnology Co., Ltd. to perform LC-MS / MS mass spectrometry detection, and detected that the FBA8 protein itself has many phosphorylation sites. Such as image 3 As shown (pep, the polypeptide containing this site is not phosphorylated; pep-Pi, the polypeptide containing this site is phosphorylated at this site; 2pep means that two polypeptides containing this site but not phosphorylated were detected by mass spectrometry , other numbers + pep and so on; 1pep-Pi means that the mass spectrometry detected 1 polypeptide containing phosphorylation at this site, other numbers + pep-Pi and so on; 2pep+1pep-Pi means that the mass spectrometry detected 2 ...

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Abstract

The invention relates to application of an FBA8 gene or FBA8 protein in preparation of a reagent with phosphate transfer activity and / or proteolytic activity, and belongs to the technical field of multifunctional enzymes. The FBA8 gene or the FBA8 protein can be used for preparing a reagent with phosphate transfer activity and / or proteolytic activity, and the aldolase activity of the FBA8 can be remarkably improved, the kinase activity of the FBA8 can be maximized, and all activities of the FBA8 can be closed; besides, the protease activity of the FBA8 can be maximized.

Description

technical field [0001] The 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 proteolysis activity. Background technique [0002] FBA8 (AT3G52930) is a fructose-1,6-bisphosphate aldolase in Arabidopsis, 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 into 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, German physiologist Warburg discovered that cancer cells consume a large amount of glucose through the glycolysis pathway compared with other adjacent tissue...

Claims

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Application Information

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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
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