A Bacterial Luciferase-Bret-Based Method for Detecting Protein Interactions

Abstract

The invention discloses a method for detection of protein-protein interaction through a BRET technology based on bacterial Luciferase. A fluorescent donor clonal pBluescript-pLac::luxAB-MCS is constructed; Green fluorescent protein (GFP) and the like are selected as acceptor proteins; appropriate enzyme cutting sites are chosen in MCS, and three kinds of fluorescent proteins are cloned into the MCS respectively; model bodies of BRET signals are generated; the fluorescence intensity of Luciferase of each of the three BRET model bodies is detected; three model bodies with high fluorescence intensity value are selected as fluorescence donors of BRET signal generation; the expression conditions of the fluorescent proteins are detected through a biological fluorescence microscope (Leica), an appropriate fluorescence acceptor is secleted; The singals of the BRET model bodies are detected through a fluorospectrophotometer. In the method, bacterial Luciferase luxAB as a fluorescence donor for detecting interaction between two proteins and eYFP as a fluorescence acceptor generate BRET signals, and BRET models are constructed. The system can detect dynamic interaction between two proteins in vivo, raise the accuracy of detection of protein-protein interaction, and is an ideal system for detection of protein-protein interaction in prokaryotic cells.

Description

technical field [0001] The invention belongs to the technical field of biological sciences, in particular to a method for detecting protein interaction based on bacterial luciferase BRET technology. Background technique [0002] Cells receive exogenous or endogenous signals, and through their unique signaling pathways, regulate the expression of their genes to maintain their biological characteristics. In this process, proteins play an important role, which can regulate and mediate cell Many biological activities of many proteins, although some proteins can function in the form of monomers, but most of the proteins work together with chaperone molecules or form complexes with other proteins. Therefore, in order to better To understand the biological activity of cells, it is necessary to have a good understanding of the functions of protein monomers and complexes, which involves the study of protein interactions. In modern molecular biology, the study of protein interactions ...

AI Technical Summary

Problems solved by technology

[0007] The purpose of the embodiments of the present invention is to provide a method for detecting protein interaction based on bacterial luciferase BRET technology, which aims to solve the existing BRET technology with complex operation, need to select a suitable fluorescent receptor, and low detection accuracy. Detecting suboptimal problems in prokaryotic cells

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