Imaging and tracking of interaction between proteins in living cells by utilizing bimolecular fluorescence complementation technology based on self-linkage label

A self-ligating, protein-based technology for use in cell biology

Inactive Publication Date: 2018-09-11
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The technical purpose of the present invention is to develop a new method for studying protein interaction based on bimolecular fluorescence complementation of self-linked tagged proteins, which can realize the purpose of long-term tracking of a pair of interacting proteins at the single-molecule level in living cells, to solve the problem of living cells. Imaging and Dynamic Observation of Interacting Protein Pairs in Cells

Method used

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  • Imaging and tracking of interaction between proteins in living cells by utilizing bimolecular fluorescence complementation technology based on self-linkage label
  • Imaging and tracking of interaction between proteins in living cells by utilizing bimolecular fluorescence complementation technology based on self-linkage label
  • Imaging and tracking of interaction between proteins in living cells by utilizing bimolecular fluorescence complementation technology based on self-linkage label

Examples

Experimental program
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Effect test

Embodiment 1

[0025] Example 1: Screening for suitable splitting sites of self-ligation tags:

[0026] (1) The requirement for the cleavage site is an irregular region between protein secondary structures, which does not affect the function of the protein. Because HaloTag is transformed from Escherichia coli dehalogenase, the crystal structure of Escherichia coli dehalogenase has been resolved but there is no crystal structure of HaloTag, so the cleavage site is designed according to the three-dimensional structure of Escherichia coli dehalogenase. Compare the protein sequences of HaloTag and E. coli dehalogenase to determine the cleavage site of HaloTag. A total of 17 split sites were designed in this experiment, namely 19, 33, 48, 58, 78, 98, 121, 141, 156, 166, 180, 207, 234, 244, 261, 269, and 278.

[0027] (2) The corresponding DNA fragments were amplified from the plasmids containing β-Fos, β-Fos(ΔZIP), and β-Jun by PCR. Add restriction enzyme sites to the amplified primers. Purifi...

Embodiment 2

[0041] Example 2: Measuring the maturation kinetics of split self-attachment tags:

[0042] (1) The corresponding DNA fragments were amplified from the plasmids containing FKBP and FRB by PCR. Add restriction enzyme sites to the amplified primers. Purified DNA fragments were recovered by DNA agarose gel electrophoresis and a gel purification kit to obtain FKBP and FRB fragments. For the PCR amplification system used, refer to step (2) of Example 1.

[0043] (2) The vectors pcDNA3.1(+)-HaloTag N-β-Jun and pcDNA3.1(+)-β-Fos-HaloTag C were digested overnight with restriction enzymes respectively to obtain the cut vector pcDNA3.1( +)-HaloTag N and pcDNA3.1(+)-HaloTagC. At the same time, the FKBP and FRB fragments were double-digested overnight with the same restriction enzymes. Pure DNA fragments were recovered by DNA agarose gel electrophoresis and gel purification kit. For the enzyme digestion system used, refer to step (3) of Example 1.

[0044] (3) pcDNA3.1(+)-HaloTag N ...

Embodiment 3

[0051] Example 3: Comparison of bimolecular fluorescence complementation of self-linked tags and photoconvertible fluorescent protein bimolecular fluorescence complementation:

[0052] (1) The corresponding mMaple3N175 and mMaple3C174 fragments were amplified from the plasmid containing mMaple3 by PCR. Add restriction enzyme sites to the amplified primers. Purified DNA fragments were recovered by DNA agarose gel electrophoresis and gel purification kit to obtain mMaple3N175 and mMaple3C174 fragments. For the PCR amplification system used, refer to step (2) of Example 1.

[0053] (2) The vectors pcDNA3.1(+)-HaloTag N-β-Jun and pcDNA3.1(+)-β-Fos-HaloTag C were digested overnight with restriction enzymes respectively to obtain the cut vector pcDNA3.1( +)-β-Jun and pcDNA3.1(+)-β-Fos. At the same time, mMaple3 N174 and mMaple3C175 fragments were double digested overnight with the same restriction enzymes. Pure DNA fragments were recovered by DNA agarose gel electrophoresis and ...

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Abstract

The invention discloses imaging and tracking of interaction between proteins in living cells by utilizing bimolecular fluorescence complementation technology based on a self-linkage label, and revealsan interaction relation between the proteins and explores that a dynamic behavior of an interaction compound is already become a hot spot for proteomics research. The interaction between proteins forms a basis of cellular activity. An existing method for researching the interaction of the proteins do not simultaneously have the characteristics of living cells, high temporal-spatial resolution, single molecules and the like, and therefore, a new method for making and tracking the interaction between the proteins needs to be developed. The invention discloses a method for novel marking the interaction compound of the proteins by adopting the bimolecular fluorescence complementation based on the self-linkage label. The self-linkage label is split into two parts at the proper site, and two proteins having the interaction effect are fused respectively, and due to the interaction of the two proteins, the split self-linkage labels are pulled spatially and closely so as to form a complete self-linkage label; by adding dyes, the interaction compound can give out fluorescent light. The novel marking method utilizing the bimolecular fluorescence complementation based on the self-linkage label is widely applied to cell biology, and single molecular horizontal detection and tracking on the interaction of the pair of proteins in the cells in a high temporal-spatial resolution manner can berealized.

Description

technical field [0001] The invention relates to a new method for detection and tracking of protein interaction in living cells and its application in cell biology. in the field of cell biology. Background technique [0002] The central dogma points out that DNA is transcribed to form RNA, and RNA is translated into protein to function, and protein is the basis of all life activities in cells. Cells can selectively receive exogenous or endogenous regulatory signals, and regulate gene expression through their unique signal regulation pathways to maintain their adaptation to the environment and their own conditions. Therefore, protein occupies a very important position, it can regulate and mediate many biological processes of cells. Although some proteins can function as monomers, the vast majority of proteins cooperate with their partners or form complex machinery with other proteins to function. Therefore, in order to better understand the various life activities of cells,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N21/64
CPCG01N21/6428G01N33/68G01N2021/6439
Inventor 邵世鹏孙超英孙育杰
Owner PEKING UNIV
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