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Fluorescent protein, and preparation method and application thereof

A fluorescent protein and fusion protein technology, applied in the field of fluorescent protein and its preparation, can solve problems such as poor fusion performance, poor photobleaching, and unfavorable construction of molecular probes

Active Publication Date: 2020-07-07
SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, CyOFP1 is a dimer with poor fusion performance, which is not conducive to its application in the construction of molecular probes, and the poor photobleaching of mCyRFP1 is not conducive to long-term imaging
In addition, although the efficiency of the existing BRET system has been improved compared to before, it still needs to be further improved

Method used

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  • Fluorescent protein, and preparation method and application thereof
  • Fluorescent protein, and preparation method and application thereof
  • Fluorescent protein, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] Example 1 Preparation and detection of fluorescent protein mCyRFP2

[0087] The red fluorescent protein CyOFP1 was subjected to site-directed mutagenesis, and then the mutants were expressed and screened on the constitutive expression vector pNCS, and the expression strain used was XL-10Gold (purchased from Agilent Technologies). In order to ensure the integrity of the library, 10 clones were set up for each mutant, and finally the fluorescent properties of the mutant were detected by visual resolution and blue LED excitation light through an orange acrylic filter, and were detected by high performance liquid chromatography HPLC (LC-20A ( SHIMADZU)) detect protein monomer, the molecular sieve column used is Superdex 200 10 / 300GL column (GE Bioscience), screen out the single clone expressing monomeric red fluorescent protein (such as figure 1 shown), the fluorescent protein was named mCyRFP2. The sequencing results show that the fluorescent protein mCyRFP2 provided in t...

Embodiment 2

[0104] Example 2 Monomer identification of fluorescent protein mCyRFP2

[0105] (1) Bacteria expressing mCyRFP2 were lysed using B-PER II (purchased from Pierce), and then HisPur Cobalt Resin (purchased from Pierce) was used to purify the protein, followed by an Econo-Pac 10DG gravity flow chromatography column (purchased from U.S. Bio-Rad company) to desalt. After completing the above protein purification steps, use Lambda35UV / VISand LS-55 fluorescence spectrometer (purchased from Perkin Elmer) to detect the absorption spectrum, excitation spectrum and emission spectrum of mCyRFP2.

[0106] The result is as figure 2 As shown, the protein sample concentration is 10mg / mL, and the flow rate is 0.5mL / min (FusionRed is the best monomeric red fluorescent protein known so far, and mKate2 is a dimer fluorescent protein known so far). The black dotted line represents mKate2, the black solid line represents CyOFP1, the gray solid line represents FusionRed, the gray dotted line repre...

Embodiment 3

[0110] Example 3 Identification of the photostability of the fluorescent protein mCyRFP2

[0111] The genes containing mCyRFP2, CyOFP1, mCyRFP1 and mNeonGreen were respectively integrated into the nucleus-localized plasmids and transfected into HeLa cells by Lipofectamine 2000 kit (purchased from GE Healthcare Dharmacon). A two-photon upright fluorescence microscope (A1MP, Nikon Instruments, numerical aperture 20×0.5) was used to excite the nuclei containing mCyRFP2, CyOFP1, mCyRFP1 and mNeonGreen proteins with 920 nm excitation light, and the excitation energy used was 10 mM. Draw a curve of fluorescence decay with the extension of excitation time, the result is as follows Figure 4 As shown in the figure, it can be seen that with the prolongation of the illumination time, the fluorescence intensity values ​​of the two proteins CyOFP and mCyRFP1 decreased rapidly, but the fluorescence intensity values ​​of the two proteins mCyRFP2 and mEGFP decreased slowly. Even after 110 sec...

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Abstract

The present invention provides a fluorescent protein, and a preparation method and an application thereof, and relates to the technical field of biology. The novel red large Stokes shift fluorescent protein is screened out, can be combined with a green fluorescent protein in a single photon and multi-photon microscopy imaging together to be effectively stimulated by monochromatic light for multi-color imaging, has good monomerity, and also has greatly improved light stability. At the same time, the fluorescent protein can be used as a donor to be combined with an acceptor luciferase to form anew highly sensitive bioluminescence resonance energy transfer (BRET) system in organisms. When detecting intramolecular interactions in cells, a dynamic range of the bioluminescence resonance energytransfer system is 2.27 times higher than that of a traditional bioluminescence resonance energy transfer system. The application of the provided protein improves efficiency of the BRET system and improves the dynamic range of the bioluminescence resonance energy transfer system for detecting the intramolecular interactions.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a fluorescent protein and its preparation method and application. Background technique [0002] With the advent of the post-genome era, as an important component of organism structure and the embodiment of biological traits, protein research has increasingly become the focus of life science research, especially in the aspect of protein-protein interaction. Traditional methods for detecting protein-protein interactions include yeast two-hybrid, immunocolocalization, tandem affinity purification, mass spectrometry identification, and co-immunoprecipitation, etc., all of which are destructive to cellular structures. Fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) overcome the limitations of traditional detection methods and can monitor intracellular protein-protein interactions in real time without damaging cells. [0003] BRET was fir...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C07K19/00C12N15/12C12Q1/66G01N21/64
CPCC07K14/43595C12Q1/66G01N21/64C07K2319/00
Inventor 储军张楚秋郭育奇邓梦颖
Owner SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI
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