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Method, vectors and kit for detection of protein interactome

A detection method and protein technology, applied in the field of systems biology research, can solve problems such as the inability to detect correlations and the inability to detect the interaction network of multiple proteins, and achieve the effect of improving throughput

Inactive Publication Date: 2018-01-26
CENT FOR EXCELLENCE IN MOLECULAR CELL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the in vivo protein interaction detection technologies widely used in the field of biological research, including yeast two-hybrid technology (Y2H), fluorescence resonance energy transfer technology (FRET) and protein fragment complementation technology (PCA), can only target cells. Therefore, researchers cannot directly detect the interaction network containing multiple proteins, and cannot detect the correlation between different protein interactions.

Method used

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  • Method, vectors and kit for detection of protein interactome
  • Method, vectors and kit for detection of protein interactome
  • Method, vectors and kit for detection of protein interactome

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0142] 1. Experimental materials and preparation

[0143] 1. Protein Interaction Footprinting (PiF)

[0144] (1) Strains and vectors

[0145] Cloned strain: Escherichia coli K12 strain DH5α (Sangon Biotech, SD8411)

[0146] Expression strain: Escherichia coli K12 strain JM109 (Takara, D9052A)

[0147] Carrier:

[0148] Based on pSP73 (Promega, P2221) and

[0149] pSB1K3 (iGEM Distribution)

[0150] culture medium

[0151] LB liquid medium:

[0152] Sodium chloride 1g, yeast extract 0.5g, peptone 1g, double distilled water 100ml

[0153] LB solid medium:

[0154] Sodium chloride 1g, yeast extract 0.5g, peptone 1g, agar 1.2g, double distilled water 100ml

[0155] M9 histidine-deficient medium:

[0156] Solution A: 2ml of 20% glucose solution, 1ml of 20mM adenine solution, 10ml of 10X histidine deficient amino acid supplement solution B: 0.1ml of 1M magnesium sulfate solution, 0.1ml of 1M thiamine hydrochloride solution, 0.1ml of 10mM zinc sulfate solution, 0.1ml of 10...

Embodiment 2

[0262] Example 2 Using PiF technology to simultaneously detect the homodimeric interaction of two different proteins in a cell

[0263] 1. Design of detection DNA sequence

[0264] In order to detect the homodimerization of two different proteins at the same time, two different proteins were constructed as fusion proteins with DNA-binding domains CI(N,wt) and CI(N,mut), so there are two DNA-binding structures in this detection system The domain dimerization combinations are CI(N,wt)CI(N,wt) and CI(N,mut)CI(N,mut). Based on this, two DNA sequences were designed and screened to specifically bind to the dimerization combinations of the above DNA-binding domains.

[0265] (1) Calculate the protein-DNA complex interaction energy

[0266] According to the steps described in the technical implementation, we downloaded the three-dimensional structure data of the CI(N,wt) dimer-DNA complex from the PDB database (PDB number: 1LMB), and used FoldX to calculate a series of DNA sequences...

Embodiment 3

[0333] Example 3 Using PiF technology to simultaneously detect three different protein interactions in a cell

[0334] 1. Design of detection DNA sequence

[0335] On the basis of Example 2, in addition to the homodimeric interactions of two proteins, we also need to simultaneously detect the heterodimeric interactions of two proteins, that is, to simultaneously detect three interactions between two proteins. Therefore, we need to design specific binding DNA sequences for the DNA-binding domain dimerization combination CI(N,wt)CI(N,mut).

[0336] (1) Calculation of protein-DNA interaction energy

[0337] Similar to Example 2, we used FoldX to calculate the interaction of a series of protein-DNA complexes containing DNA sequence point mutations compared with wild-type based on the structural data of the DNA-binding domain heterodimer-DNA complex can change (such as Figure 14 shown).

[0338] (2) Design of core sequence

[0339] Based on the calculation results of the abov...

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Abstract

The invention specifically relates to a method for simultaneous quantitative detection of a protein interactome formed by interactions among a plurality of different proteins in a single cell, and vectors and a kit used therein. The vectors and the kit used in the method are based on protein-interactome footprinting. According to the invention, simultaneous detection of interactions among a plurality of different proteins in a same cell is realized; the detection flux of protein interaction is greatly improved; and an approach is provided for direct detection of interactions among proteins andrelevance of the interactions of protein interactomes at a system level.

Description

technical field [0001] The invention belongs to the technical field of systems biology research, and specifically relates to a method for quantitatively detecting protein interactomes formed by the interaction of multiple different proteins in a single cell, a used carrier and a kit. Background technique [0002] More than 80% of proteins in cells function in the form of protein complexes, and various proteins form a complex protein interactome (Interactome) through extensive interactions. Among them, there is a certain mutual influence between different protein interactions, that is to say, the correlation between protein interactions may play an important role in the protein interactome. However, the in vivo protein interaction detection technologies widely used in the field of biological research, including yeast two-hybrid technology (Y2H), fluorescence resonance energy transfer technology (FRET) and protein fragment complementation technology (PCA), can only target cell...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6818C12N15/70G01N33/68
Inventor 吴家睿梁治骆斯伟
Owner CENT FOR EXCELLENCE IN MOLECULAR CELL SCI CHINESE ACAD OF SCI