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Method for effectively screening membrane protein interacting protein based on yeast two-hybrid technology

A yeast two-hybrid, membrane protein technology, applied in the field of biomolecules, can solve problems such as affinity reduction

Inactive Publication Date: 2019-11-26
BEIJING FORESTRY UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Replace the 13th isoleucine of NubI with alanine or glycine, and the affinity between the mutated NubG and Cub is greatly reduced

Method used

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  • Method for effectively screening membrane protein interacting protein based on yeast two-hybrid technology
  • Method for effectively screening membrane protein interacting protein based on yeast two-hybrid technology
  • Method for effectively screening membrane protein interacting protein based on yeast two-hybrid technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0078] Take Populus trichocarpa Potri.001G190400.1 as an implementation case

[0079] Step 1: Analyze the structure of the bait protein, design primers, and use Sfi1 enzyme as the restriction site to design primers to construct the bait vector.

[0080] pBT3-N:F ATTAACAAG GCC ATT ACG GCCATGGAGAAGAAGGTGGTGGT R TTA AAG ATACAG TGA ATG TGA GGCCGCCTCGGCCAATCAGTT

[0081] pBT3-SUC:F ATTAACAAG GCC ATT ACGGCCATGGAGAAGAAGGTGGTGGT RTTAAAGATACAGTGAATGTGAGGGGCCGCCTCGGCCAATCAGTT

[0082] pBT3-STE:F ATTAACAAG GCC ATT ACG GCCATGGAGAAGAAGGTGGTGGT

[0083] R TTAAAGATACAGTGAATGTGAGGGGCCGCCTCGGCCAATCAGTT

[0084] Step 2, self-activation detection and bait carrier function verification, using plasmids pTSU2-APP and pPR3-N as negative controls, and plasmids pTSU2-APP and pNubG-Fe65 as positive controls.

[0085] 1) Streak the NMY51 yeast strain on the YPDA plate, and culture it upside down at 30°C for about 2 days;

[0086] 2) Pick several yeast clones with a pipette tip and place them in 50ml...

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Abstract

The invention relates to a method for screening membrane protein interacting protein based on a yeast two-hybrid technology. Through the steps of bait protein carrier selection, self-activation detection, bait carrier function verification, membrane system two-hybrid library screening yeast, plasmid extraction, yeast plasmid propagation and the like, the selection of bait carriers is optimized, and by selecting suitable bait carriers, more interacting proteins can be screened to the maximum extent; the preparation process of yeast competent cells is optimized, the bacterium shaking process iswell grasped by a method close to certain quantity, thus the state of the yeast competent cells is optimized, the expression of a library is facilitated, the experiment progress can be well grasped, and the experiment stability is improved; and according to the final PCR verification of the membrane protein interaction based on an ubiquitin system, the efficiency and stability of a membrane protein interaction research method are greatly improved, and advanced technical means and theoretical basis are provided for science researchers.

Description

technical field [0001] The invention relates to the field of biomolecules, in particular to a method for efficiently screening membrane protein interaction proteins by using yeast two-hybrid. Background technique [0002] Yeast two-hybrid is a fast and efficient method for analyzing protein interactions. According to different detection principles and objects, the yeast two-hybrid system can be divided into two categories: GAL4 (gene of yeast transcriptional activator GAL4) system and ubiquitin system. The GAL4 system is used to study the interaction of nuclear proteins, while the yeast two-hybrid technology based on the ubiquitin system can specifically study the interaction of membrane proteins, providing membrane protein researchers with a new technical means of in vivo interaction. In addition, the construction of a membrane protein capture library and the use of bait membrane proteins for interaction experiments can efficiently screen the target proteins that interact ...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12N15/81C12Q1/6876
CPCC12N15/10C12N15/81C12Q1/6876
Inventor 林金星郑文清张曦冯圣年王树芳祝蕾毋瑞华
Owner BEIJING FORESTRY UNIVERSITY
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