Protein interaction detection method with low false positive rate

A technology of false positive rate and detection method, which is applied in the field of molecular biology, can solve the problems affecting the application of BiFC technology and the difficulty of controlling the ratio of different gene expression, and achieve the effect of simple operation, reducing false positive and expanding the scope of application

Active Publication Date: 2013-09-11
HUAZHONG UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Obviously, in the process of multi-plasmid transfection, the expression ratio of different genes is difficult to control
This problem greatly affects the application of BiFC technology in the detection of multiple pairs of protein interactions.

Method used

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  • Protein interaction detection method with low false positive rate
  • Protein interaction detection method with low false positive rate
  • Protein interaction detection method with low false positive rate

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Experimental program
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Effect test

Embodiment 1

[0044] This implementation example uses the mLumin-BiFC system based on dual expression vectors to establish a protein interaction detection method with a low false positive rate. Based on this purpose, bFos was connected to the N-terminal of the mLumin protein sequence (hereinafter referred to as LN), and bJun was connected to the C-terminal of the mLumin protein sequence (hereinafter referred to as LC).

[0045] refer to figure 1 , figure 1 It is the structural diagram of the BiFC molecular probe based on the mLumin155 site, which consists of the positive group mLumin-Ln155-bFos / mLumin-Lc155-bJun and the negative group mLumin-Ln55-ΔbFos / mLumin-Lc155-bJun of the probe. Among them, ΔbFos is the bFos after deletion mutation, which cannot interact with bJun under physiological conditions, and thus does not generate BiFC fluorescence signal.

[0046] The specific steps are as follows: firstly, pBud-Ln-Lc is constructed by applying genetic engineering technology, and the plasmid...

Embodiment 2

[0051] Raf1 is the main effector protein of KRas, an important component of cell signaling pathway, and plays an important role in growth, development and carcinogenesis. The Ras-binding domain RBD of Kras-Raf1 protein has been used as a specific probe of Ras activity in many studies. GTP-bound KRas has a stronger affinity for RBD than GDP-bound KRas, and the affinity between GDP-bound Ras and Raf1 is too weak to participate in signal transduction. Therefore, heterodimerization of RBD with KRas may serve as a good protein interaction model for the development of PPI detection methods in living cells.

[0052] Figure 8 to Figure 10 It is the fluorescence map of COS-7 cells transfected with pBud-Ln-RBD-Lc-KRas, pBud-Ln-RBD-Lc-KRas 12v and pBud-Ln-RBD-Lc-KrasC185S. RBD and KRas or KRas mutants were inserted into the pBud-Ln-Lc vector to construct three groups of pBud-Ln-RBD-Lc-KRas, pBud-Ln-RBD-Lc-KRas12v and pBud-Ln-RBD-Lc-KrasC185S probe. After the COS-7 cells were transfe...

Embodiment 3

[0054] Grb2 is an important adapter protein in the cell signaling pathway. Through Grb2, KRas can interact with different signaling pathway downstream proteins. The activation of KRas is mainly accomplished through its interaction with the Grb2-SOS1 protein complex.

[0055] The fusion fragments Grb2 and KRas or their mutants were cloned into pBud-Ln-Lc to construct pBud-Ln-Grb2-Lc-KRas, pBud-Ln-Grb2-Lc-KRas 12v and pBud-Ln-Grb2-Lc-KRas185S . After the plasmids were transfected into COS-7 cells, they were cultured at 37°C for 24 hours before imaging. refer to Figure 11 to Figure 13 , the red fluorescence of cells expressing pBud-Ln-Grb2-Lc-KRas12v on the cell membrane was significantly stronger than that of pBud-Ln-Grb2-Lc-KRas, but no obvious fluorescence was seen in pBud-Ln-Grb2-Lc-KRas185S The fluorescence indicates that the BiFC probe can be applied to the detection of complexes formed by more than two proteins, and also verifies that the activation of KRas enhances th...

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Abstract

The invention provides a protein interaction detection method with low false positive rate, relates to a detection system and belongs to the field of a molecular biological technology. According to the method, two generally needed bimolecular fluorescence complementation (BiFC) detection systems based on single expression plasmid vectors are built in a dual expression plasmid vector system by a BiFC technology based on fluorescent protein, so the false positive rate of the BiFC detection method in research on protein-protein interaction can be reduced obviously, and quantitative analysis can be realized. The method also can be applied to screening research on unknown protein-protein interaction based on a gene library and detection on protein-protein interaction in living animals.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, in particular to a protein interaction detection method with a low false positive rate. Background technique [0002] The cell itself is the basic unit of an ordered life activity formed by the interaction of different biomolecules. The occurrence and regulation of major cell life activities are realized through the interaction between biomacromolecules (protein-protein, protein-nucleic acid, etc.). Protein-protein interaction (hereinafter referred to as "PPI") not only controls gene transcription, cell division and cell proliferation, but also mediates signal transduction, carcinogenesis transformation and adjustment in the process of cell life activities, so Studying protein-protein interactions has important life science significance. [0003] At present, for mammalian cells, there are only Fluorescence Resonance Energy Transfer (FRET) technology and Bimolecular Fluorescence Comple...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/02G01N21/64
Inventor 骆清铭张智红李向勇潘少涛
Owner HUAZHONG UNIV OF SCI & TECH
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