Method for detecting interaction between biofilm proteins and complete set of reagents used by method

A protein and biofilm technology, applied in biological testing, using carriers to introduce foreign genetic material, material inspection products, etc., can solve problems such as excessive false positive results, affecting the effect of immunoprecipitation, and affecting the results of activity experiments

Active Publication Date: 2020-10-30
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with the interaction between proteins in the cytoplasm, the detection of protein interactions on the cell membrane and organelle membranes faces greater challenges, including the difficulty of extracting proteins on the membrane structure, short interaction time and weak force, etc. Membrane protein interactions are often difficult to detect using traditional methods
For example, the classic immunoprecipitation technique is suitable for non-transmembrane proteins. After expression and purification of transmembrane proteins, the spatial structure of the transmembrane proteins will change due to separation from the membrane environment, which will affect their protein functions and further affect the effect of immunoprecipitation.
The isolated ubiquitin yeast two-hybrid (DUALmembrane) system, which aims to study the interaction of membrane proteins, requires follow-up tedious identification work due to the high number of false positive results
The protein proximity catalytic labeling technology developed in recent years, represented by BioID, APEX and PUP-IT, can identify the interaction between membrane proteins by fusing the enzyme with the function of proximity labeling to the bait protein. However, this technology exists A common problem with enzymes is that self-modification of enzymes is unavoidable, and this self-modification may affect experimental results by inhibiting enzyme activity or depleting substrates

Method used

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  • Method for detecting interaction between biofilm proteins and complete set of reagents used by method
  • Method for detecting interaction between biofilm proteins and complete set of reagents used by method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0146] Example 1, identification of cell membrane protein interaction promoting factors

[0147] 1. Preparation of recombinant vector

[0148] 1. A recombinant vector expressing the fusion protein of mCherry and FRB

[0149] The DNA fragment (comprising the recognition sequence of NcoI and XhoI) between the NcoI and XhoI recognition sequences of the pRSFDuet-1 vector (Novagen product of Merck) is replaced with the DNA molecule shown in the 11th-831th position of sequence 2 in the sequence listing, The recombinant vector pRSFDuet-1-mCherry is obtained, and the pRSFDuet-1-mCherry can express the protein shown in Sequence 1 (mCherry is fused with His-tag, denoted as His-mCherry).

[0150] Among them, the 13th-825th position of sequence 2 encodes His-mCherry shown in sequence 1, the 815-820th and 826-831st positions of sequence 2 are the recognition sequences of NcoI and XhoI respectively, and the 3rd-8th position of sequence 1 The position is the amino acid sequence of His-tag,...

Embodiment 2

[0201] Example 2, verification of cell membrane protein interaction inhibitors

[0202] 1. Preparation of recombinant vector

[0203] 1. A recombinant vector expressing the fusion protein of mCherry and KKETPV

[0204] The DNA fragment between the NcoI and XhoI recognition sequences of the pRSFDuet-1-mCherry vector of Example 1 (comprising the recognition sequences of NcoI and XhoI) was replaced with the DNA molecule shown in sequence 8 to obtain the recombinant vector pRSFDuet-1-mCherry-KKETPV , pRSFDuet-1-mCherry-KKETPV expresses the fusion protein of His-mCherry and KKETPV shown in Sequence 7 (the fusion protein is designated as mCherry-KKETPV, and contains a His tag).

[0205] 2. A recombinant vector expressing the fusion protein of GFP-LCD and FRB

[0206]The DNA fragment between the NcoI and XhoI recognition sequences of the pRSFDuet-1-GFP-LCD vector of Example 1 (comprising the recognition sequences of NcoI and XhoI) was replaced with the DNA molecule shown in sequenc...

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Abstract

The invention discloses a method for detecting interaction between biofilm proteins and a complete set of reagents used by the method. The method for detecting the interaction between biofilm proteinsdisclosed by the invention comprises the following steps of: connecting proteins X and Y to obtain X-Y; connecting proteins YL and R to obtain YL-R, wherein Y and YL interact with each other, and R can drive phase separation to generate phase change liquid drops; connecting XL with a reporter group J to obtain XL-J; introducing an X-Y encoding gene into a biological cell to express the X-Y encoding gene to obtain reconstitution cells, wherein the X-Y is positioned on a biological membrane; adding the YL-R and the XL-J into a culture system of the reconstitution cells, detecting whether signals of the J are gathered in the phase change liquid drops formed by the R or not, and determining whether interaction exists between the X and the XL or not. The method is simple and convenient to operate, high in sensitivity, low in cost and wide in applicability.

Description

technical field [0001] The invention relates to a method for detecting the interaction between biomembrane proteins and a set of reagents used in the field of biotechnology. Background technique [0002] "Phase change" as a characteristic of matter has long been known in the physics world and in daily life. In recent years, scientists have gradually discovered that the mechanism of phase change (or phase separation) also widely exists in biological cells and is used in cell life activities. important biological functions. [0003] Relevant studies have found that biomacromolecules with specific structures can be highly aggregated due to interactions at a certain concentration, and thus separated from the general solution phase to form an independent phase enriched in macromolecules (called the second phase, referred to as Different from the original solution phase), this phenomenon is called "phase transition" (or "phase separation"). Under the microscope, it can be seen t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68C12N15/70C12N1/21C12N15/85
CPCG01N33/68C12N15/70C12N15/85
Inventor 李丕龙李茹王静
Owner TSINGHUA UNIV
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