High throughput test method for SUMO modification substrate in living cells and application thereof

Abstract

The invention discloses a high throughput test method for SUMO modification substrate in living cells and application thereof. According to the method, on the basis of a BiFC system, a reverse transcription expression vector of YFPn connected with C terminals or N terminals of SUMO1 and SUMO2 / 3 is established, and cDNA of multiple genes of an object to be detected is cloned into the reverse transcription expression vector containing YFPc; fusion protein: SUMO-YFPn and protein to be screened: YFPc will be expressed stably simultaneously after virus infection. fluorescence signals can be observed under FITC wave length if the protein is reacted, and accordingly SUMO modification substrate in living cells can be screened out. According to the method, protein substrates subjected to covalent modification and non-covalent modification of SUMO1 / 2 / 3 can be screened, the combined condition of SUMO covalent or non-covalent modification substrates under stimulation and non stimulation can be screened, SUMO weakly combined protein substrates can be screened, the signal to noise ratio and the sensitivity are high, and the false positive is low.

Description

technical field [0001] The invention belongs to the technical field of protein research. More specifically, it relates to a method for high-throughput detection of SUMO-modified substrates in living cells and its application. Background technique [0002] Small Ubiquitin-like Modifier (Small Ubiquitin-like Modifier, SUMO) is a small molecular polypeptide that can modify other proteins through isopeptide bonds. SUMOylation is an important way of protein post-translational modification, which is similar to that found in animals. Protein modification by ubiquitin. SUMO molecules are highly conserved in evolution and widely exist in protozoa, metazoans, plants and fungi. Four mammalian SUMO molecules have been found, namely SUMO1, SUMO2, SUMO3 and SUMO4. Among them, the amino acid sequences of SUMO2 and SUMO3 Very close, often written as SUMO2 / 3, SUMO4 is the fourth newly discovered SUMO molecule. [0003] Under the action of SUMO pathway enzymes, SUMO molecules can be linked...

AI Technical Summary

Problems solved by technology

[0005] The technical problem to be solved by the present invention is to overcome the defect of insufficient sensitivity of the existing identification of SUMO covalently modified substrates, and to make up for the gap in the identification of SUMO non-covalently modified substrates, to provide a method that can simultaneously detect specific protein covalent and non-covalent The detection method of covalently modified substrates is used to screen the protein network that interacts with the target protein under stimulating or non-stimulating conditions, and to screen the binding of SUMO covalently or non-covalently modified substrates under stimulating and non-stimulating conditions. Screen protein substrates with weak SUMO binding, high signal-to-noise ratio sensitivity, and low false positives

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