Construction method of split-TEV-Fast system and application of split-TEV-Fast system in detecting protein interaction

A split-tev-fast and tev-fast technology, applied in the fields of biotechnology and molecular biology, can solve the problems of low reaction sensitivity, inability to accurately quantify the strength of protein interaction, and large interaction space between enzymes and substrates. The effect of sensitivity
CN108048480AActive Publication Date: 2018-05-18HUBEI UNIV
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Patent Information

Authority / Receiving Office
CN · China
Current Assignee / Owner
HUBEI UNIV
Publication Date
2018-05-18

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Abstract

The invention discloses a method for constructing a split-TEV-Fast system based on saccharomyces cerevisiae and an application of the split-TEV-Fast system in detecting a protein interaction. The method comprises the following steps: 1) constructing a vector pESD-PPI, wherein the vector comprises a forward promoter and a bidirectional promoter induced by galactose, the forward promoter is used forexpressing a TEV-Fast protease substrate as well as FLAG and HA tag sequences, and the bidirectional promoter is used for expressing resolving TEV-Fast protease and a to-be-detected target protein; 2) simultaneously inducing the promoters via the galactose, and inducing equivalent expression of a downstream gene; 3) marking cells via fluorescent antibodies which recognize the FLAG and HA tag sequences; and 4) detecting fluorescence signals on the surfaces of the cells via a flow cytometry, and judging the intensity of the protein interaction in accordance with the single and double fluorescence signals. The method provided by the invention, by adopting endoplasmic reticulum retention signal peptide differing in intensity at -COOH terminals of the TEV-Fast protease and the substrate thereof, can widen a range of detecting the protein interaction; and the intensity of the protein interaction can be analyzed depending on a finally detected proportion of single and double fluorescence signal intensities.
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Description

Technical field

[0001] The invention belongs to the technical field of biotechnology and molecular biology, and particularly relates to a method for constructing a split-TEV-Fast system and its application in detecting protein-protein interaction. Background technique

[0002] The study of protein-protein interaction has extremely important value in many biological fields. In organisms, a protein usually interacts with other proteins to further perform its function, which is the main way of biological information regulation. Protein-protein interaction clarifies the physiological and biochemical reactions and molecular mechanisms in the entire cell, and even the cellular The whole life process. At present, methods for verifying protein-protein interactions mainly include several classic experimental techniques: yeast two-hybrid technique, co-immunoprecipitation technique (Co-IP), GST pull down technique and so on. Yeast two-hybrid technology is characterized by sensitivity, hig...

Claims

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