Method for discovering membrane protein based on membrane system-yeast two-hybrid

Abstract

The invention relates to the technical field of membrane protein experiments, and discloses a method for discovering membrane protein based on membrane system-yeast two-hybrid. Comprising the following material bait cloning: pBT3-ST-A, a bait carrier: pBT3-STE, library yeast plasmids: a grass membrane library, a Clontech yeast two-hybrid system, a culture medium and a stock solution. The invention discloses a method for discovering membrane protein based on membrane system-yeast two-hybrid. A bait plasmid is transformed into recipient bacteria NMY51, and the steps of self-activation detection, library screening, library screening results, yeast positive clone identification and sequencing comparison and sequencing comparison results, yeast positive clone identification and sequencing comparison and sequencing comparison results, yeast positive clone rotation verification and the like are performed. 34 yeast positive clones obtained through screening can grow on SD-TL, SD-TLH + 30mM 3AT and SD-TLHA + 30mM 3AT defective plates through yeast positive clone rotation verification, so that the final membrane protein is obtained, and the problem that the protein acting with pBT3-ST-A is not easy to find is solved.

Description

technical field [0001] The invention relates to the technical field of membrane protein experiments, in particular to a method for discovering membrane proteins based on membrane system-yeast two-hybridization. Background technique [0002] The proteins contained in biofilms are called membrane proteins, which are the main bearers of the function of biofilms. According to the difficulty of protein separation and the location of its distribution in the membrane, membrane proteins can be basically divided into three categories: external membrane proteins or peripheral membrane proteins, internal membrane proteins or integral membrane proteins and lipid-anchored proteins, membrane proteins. Including glycoproteins, carrier proteins and enzymes, etc. Usually, some sugars are attached to the outside of the membrane protein. These sugars are equivalent to transmitting signals into the cell through the molecular structure change of the sugar itself. Techniques to study the structu...

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Problems solved by technology

[0004] Aiming at the deficiencies of the prior art, the present invention provides a method for discovering membrane proteins based on the membrane system-yeast two-hybrid, which uses the constructed pBT3-STE-A gene as bait to screen the yeast two-hybrid turf library. Multiple reporter gene detection, DNA sequencing and BLAST comparison analysis of positive clones to determine the advantages of proteins interacting with pBT3-STE-A, and solve the problem that the proteins interacting with pBT3-STE-A are not easy to find and affect the experiment

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