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Yeast transformant for screening protein with interaction with G protein beta-gamma dimer and screening method of yeast transformant

A dimer, G protein technology, applied in the field of genetic biology, can solve the problem that the function of βγ dimer cannot be fully reflected

Inactive Publication Date: 2018-09-21
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In order to screen and identify the downstream signaling elements of βγ dimer, researchers usually identify proteins that interact with β and γ subunits alone, which cannot fully reflect the function of βγ dimer
There are not many reports on large-scale screening of interacting proteins of protein dimers such as βγ dimers

Method used

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  • Yeast transformant for screening protein with interaction with G protein beta-gamma dimer and screening method of yeast transformant
  • Yeast transformant for screening protein with interaction with G protein beta-gamma dimer and screening method of yeast transformant
  • Yeast transformant for screening protein with interaction with G protein beta-gamma dimer and screening method of yeast transformant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1. Yeast transformants for screening proteins interacting with G protein βγ1 dimers

[0026] A method for constructing a yeast transformant for screening proteins interacting with G protein βγ1 dimers, comprising the steps of:

[0027] 1) Insert the coding gene (Genbank accession number: KX099865) of the Mulberry β subunit between the EcoR I and Sal I restriction sites of MCS I of the pBridge vector, and the coding gene (Genbank accession number: KX099866) of the γ1 subunit ) was inserted between the Not I and Bgl II restriction sites of the MCS II of the pBridge vector to recombine the plasmid pBridge-βγ1.

[0028] 2) Transforming the recombinant plasmid pBridge-βγ1 obtained in step 1) into AH109 yeast to obtain an AH109 yeast transformant containing the pBridge-Gβγ1 bait plasmid.

Embodiment 2

[0029] Example 2, Yeast three-hybrid screening method for interacting proteins with Morus alba βγ1

[0030] A method for yeast three-hybrid screening of proteins interacting with Morus alba βγ1, comprising the steps of:

[0031] 1) Treat 15d mulberry seedlings with a mass fraction of 0.6% NaCl, and use the Trizol method to extract and isolate RNA ( figure 1 , A and B), the first and second strands of cDNA were synthesized, and then transferred to the linearized pGADT7 vector to obtain the library plasmid, and finally the identification of the library capacity and the size of the insert were carried out ( figure 1 , C), the library capacity is greater than 3×10 6 CFUml -1 , the average insert fragment is greater than 1200bp, the positive rate is 100%, and the library index is qualified;

[0032] 2) The recombinant plasmids pBridge-βγ1 and pGADT7 were co-transformed into AH109 yeast, 6 colonies were randomly selected for self-activation detection, and the detection of the HIS...

Embodiment 3

[0038] Example 3, Screening Yeast Transformants for Proteins Interacting with G Protein βγ2 Dimers

[0039] A method for constructing a yeast transformant for screening proteins interacting with G protein βγ2 dimers, comprising the steps of:

[0040] 1) Insert the coding gene (Genbank accession number: KX099865) of the Mulberry β subunit between the EcoR I and Sal I restriction sites of MCS I of the pBridge vector, and the coding gene (Genbank accession number: KX099867 ) were respectively inserted between the Not I and Bgl II restriction sites of MCS II of the pBridge vector to recombine the plasmid pBridge-βγ2.

[0041] 2) Transform the recombinant plasmid pBridge-βγ2 obtained in step 1) into AH109 yeast to obtain an AH109 yeast transformant containing the pBridge-Gβγ2 bait plasmid.

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Abstract

The invention relates to yeast transformant for screening a protein with interaction with G protein beta-gamma dimer and a screening method of the yeast transformant. Yeast transformant has encoding genes of a beta-subunit and a gamma-subunit of mulberry, a mulberry cDNA (Complementary Deoxyribonucleic Acid) library is converted into the yeast transformant capable of simultaneously expressing thebeta-subunit and the gamma-subunit, two types of nutrition defect screening of His and Met are carried out to obtain positive cloning with interaction with beta-gamma dimer, and finally the protein with interaction with the G protein beta-gamma dimer of the mulberry can be successfully screened. By adopting the method, a protein dimer interaction protein can be efficiently screened, and the methodcan be successfully applied to screening on a mulberry G protein beta-gamma dimer interaction protein.

Description

technical field [0001] The invention belongs to the technical field of genetic biology, and relates to a yeast transformant for screening proteins interacting with G protein beta gamma dimers; and also relates to a screening method and application. Background technique [0002] Protein-protein interaction refers to the function of two or more protein molecules forming a protein complex through non-covalent bonds. This process is a major component of the cell biochemical reaction network. The methods currently used to study protein interactions include yeast two-hybrid system, phage display technology, plasmon resonance technology, fluorescence energy transfer technology, antibody and protein array technology, co-immunoprecipitation technology and pull-down technology. Among them, the yeast two-hybrid system is the most rapid and direct method for protein interaction research. The basic principle is that a complete yeast transcription factor GAL4 can be divided into two funct...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81G01N33/68
CPCG01N33/68C12N15/81C07K14/415
Inventor 赵爱春刘长英龙定沛胡杰向仲怀
Owner SOUTHWEST UNIVERSITY
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