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LRR (leucine-rich repeat) receptor kinase substrate searching method

A receptor kinase and substrate technology, applied in the field of finding substrates for LRR receptor kinases, can solve the problems of difficult purification of LRR receptor kinases, difficulty in finding direct downstream substrates, low yield, etc.

Active Publication Date: 2018-10-26
INST OF FORESTRY CHINESE ACAD OF FORESTRY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Another method to find direct effectors downstream of LRR receptor kinase is yeast two-hybrid, but the low yield and false positives will also limit the use of this method
Additionally, as membrane proteins, LRR receptor kinases are often difficult to purify
This also makes it difficult for conventional biochemical strategies to find its immediate downstream substrates

Method used

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  • LRR (leucine-rich repeat) receptor kinase substrate searching method
  • LRR (leucine-rich repeat) receptor kinase substrate searching method
  • LRR (leucine-rich repeat) receptor kinase substrate searching method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0154] Pretreatment of protein G agarose:

[0155] (1) Take 640 μl of protein G agarose (Protein G agarose beads produced by Beyond Company, 2ml specification, product number is P2009), and divide into two equal parts. Centrifuge at 13000rpm, 4°C for 15s, discard the supernatant;

[0156] (2) Resuspend protein G agarose with 1.3ml of the first phosphate buffer (0.1M KH2PO4, 0.1M K2HPO4, pH 7.5), centrifuge at 13000rpm, 4°C for 15s, discard the supernatant, and repeat three times.

[0157] (3) Add 540 μl of the first phosphate buffer to the protein G agarose with a volume of about 100 μl in step (2), the total volume is about 640 μl;

[0158] (4) After mixing the suspension in step (3), pipette 200 μl of the suspension from one tube and add it to another tube. The smaller tube (about 440 μl) is used to remove non-specificity before immunoprecipitation Binding protein; one larger tube (approximately 840 μl) for later immunoprecipitation. Both tubes were placed at 4°C for late...

Embodiment 2

[0160] Anti-phosphorothioate monoclonal antibody conjugated to protein G agarose:

[0161] (1) Prepare four 1.5ml centrifuge tubes, add 788μl deionized water and 132μl second phosphate buffer (1MKH 2 PO 4 , 1M K 2 HPO 4 , pH 7.5);

[0162] (2) Take out 200 μl from the large tube of protein G agarose processed in Example 1 and add them to four new 1.5ml EP tubes, and name them as A, B, C, and D respectively;

[0163] (3) Centrifuge the four EP tubes in step (2) at 13000rpm, 4°C for 15s, and discard the supernatant. Then add the solution in step (1) one-to-one to A, B, C, and D tubes respectively, and mix well;

[0164] (4) Add 4 μl (1 μg / μl) of anti-phosphorothioate monoclonal antibody (manufactured by ABcam, 50 μl size, catalog number ab92570) to each tube of step (3). After mixing, shake gently at 4°C for 5 hours;

[0165] (5) Centrifuge each tube in step (4) at 13,000 rpm at 4°C for 15 seconds, and discard the supernatant. After resuspending with 1.3ml of boric acid ...

Embodiment 3

[0171] Substrate search for LRR receptor kinase-PXY:

[0172] S1: Incubate PXY with ATP-γ-S to prepare the first mixture:

[0173] (S11) Take four 1.5ml EP tubes and name them as A1, B1, C1, D1 respectively, and the C1 and D1 tubes are used as the control group.

[0174] (S12) Add 20 μl 10 μM PXY, 8 μl 10 mM CLE41 (sequence HEV Hy PSG Hy PNPISN; Hy P, means 4-hydroxyproline, synthesized by Shanghai Sangong Company), 1μl 1mM ATP-γ-S and 21μl kinase buffer (25mM Tris-HCl, pH 7.5, 0.5mM DTT, 10mM MgCl2) (containing 0.225mM dodecyl -β-D-maltoside);

[0175] (S13) Add 20 μl 10 μM PXY, 1 μl 1 mM ATP-γ-S (manufactured by ABcam Company, 1 mg specification, product number is ab138911) and 29 μl kinase buffer (containing 0.225 mM dodecyl-β-D-maltose) to tube B1 glycosides);

[0176] (S14) Add 1 μl 1 mM ATP-γ-S and 49 μl kinase buffer (containing 0.225 mM dodecyl-β-D-maltoside) to C1 tube;

[0177] (S15) Add 50 μl of kinase buffer (containing 0.225 mM dodecyl-β-D-maltoside) to tub...

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Abstract

The invention provides an LRR (leucine-rich repeat) receptor kinase substrate searching method. The LRR receptor kinase substrate searching method includes: S1, incubating LRR receptor kinase along with ATP-gamma-S to obtain a first mixture; S2, incubating the first mixture along with total protein which is rich in LRR receptor kinase structures so as to obtain a second mixture; S3, incubating thesecond mixture along with PNBM to obtain a third mixture; S4, removing the PNBM in the third mixture to obtain a fourth mixture; S5, incubating the fourth mixture with pretreated protein G agarose toobtain first supernatant; S6, incubating the first supernatant along with a phosphorothioate resistant monoclonal antibody, which is in coupled linkage with the protein G agarose, so as to obtain first sediment; S7, analyzing the first sediment to determine an LRR receptor kinase substrate.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for finding a substrate of LRR receptor kinase. Background technique [0002] LRR receptor kinase is a transmembrane protein expressed on the surface of plant cell membranes. As an important class of regulatory molecules affecting plant growth and development, LRR receptor kinases widely act on various signal transduction processes. [0003] In the process of studying LRR receptor kinases, the acquisition of its downstream effectors is usually carried out by mutating transgenic plants under the background of overexpressing target genes. The protein corresponding to the mutant gene contained in the phenotype-suppressing mutant is the downstream target of the receptor kinase. Another method for finding direct effectors downstream of LRR receptor kinases is yeast two-hybrid, but the low yield and false positives also limit the use of this method. Additionally, as...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/573
CPCG01N33/573G01N33/577G01N2333/91215
Inventor 张建国国鹏王兆山
Owner INST OF FORESTRY CHINESE ACAD OF FORESTRY
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