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Large stokes displacement fluorescent protein CyOFP and application thereof

A fluorescent protein and Stokes technology, applied in the field of biomedical optics and molecular imaging, can solve the problems of unsatisfactory two-photon synchronous imaging BRET system, low sensitivity of two-photon imaging, shallow depth of in vivo imaging, etc., to achieve Effect of improved penetration, improved depth and sensitivity, strong fluorescent signal and sensitivity

Inactive Publication Date: 2016-04-06
SHENZHEN INST OF ADVANCED TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] However, in living organisms, the BRET system for two-photon simultaneous imaging is not ideal
The classic BRET technology uses mostly green fluorescent protein mutants as receptors, the system emits short light, and the in vivo imaging depth is shallow; the existing large Stokes shift fluorescent protein cannot be efficiently excited together with green fluorescent protein, and the emission Produces photons different from EGFP, so the sensitivity of two-photon imaging is relatively low

Method used

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  • Large stokes displacement fluorescent protein CyOFP and application thereof
  • Large stokes displacement fluorescent protein CyOFP and application thereof
  • Large stokes displacement fluorescent protein CyOFP and application thereof

Examples

Experimental program
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Embodiment 1

[0055] This embodiment provides a large Stokes shift fluorescent protein CyOFP, which is obtained by site-directed mutagenesis screening on the basis of the far-infrared fluorescent protein mNeptune, and its acquisition process is as follows:

[0056] Carry out site-directed mutagenesis to the far-infrared fluorescent protein mNeptune, wherein the 1-240th positions of the amino acid sequence of mNeptune correspond to the 4th-244th positions shown in SEQIDNo: 1; then carry out the mutant on the constitutive expression vector pNCS For expression and screening, 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, which was finally distinguished by naked eyes and blue LED excitation light through an orange acrylic filter Detect the fluorescent properties of the mutant, and screen out a single clone expressing a fluorescent protein with a large Stokes shift. The fluores...

Embodiment 2

[0061] Example 2 Functional experiments corresponding to mutation sites

[0062] (1) Experiments embodying Stokes shift

[0063] The mNeptune was randomly mutated and screened by error-prone PCR method, and the bacterial single clone with an orange color was selected. Through sequencing, the fluorescent protein with only the M160K mutation site was screened out, which was mNeptune+M160K.

[0064] Take 150 microliters of purified protein and place it in a black 96-well plate, and measure the excitation and emission spectra using a SafireII multifunctional microplate reader from Tecan Company. Such as image 3 as shown, image 3 For this example fluorescent protein "mNeptune+M160K" and mNeptune excitation and emission spectrum comparison chart, image 3 The picture in the upper right corner of the curve is a white light picture of bacteria expressing mNeptune and mNeptune-M160K.

[0065] Depend on image 3 It can be seen that M160K can cause the excitation and emission of m...

Embodiment 3

[0078] Embodiment 3CyOFP excitation spectrum and emission spectrum test experiment

[0079] B-PERII (purchased from Pierce Company) was used to lyse the bacterial strain expressing CyOFP in the above example 1, and then HisPurCobaltResin (purchased from Pierce Company) was used to purify the protein, followed by an Econo-Pac10DG gravity flow chromatography column (purchased from Bio -Rad) to salt. After completing the above protein purification steps, use a Lambda35UV / VISandLS-55 fluorescence spectrometer (purchased from PerkinElmer) to detect the excitation and emission spectra of CyOFP, as Figure 7 shown; in addition, the comparison between CyOFP and some red fluorescent proteins in terms of quantum yield is shown in Table 3.

[0080] table 3

[0081]

[0082]

[0083] Experimental results show that: CyOFP has a wide range of excitation spectrum, and can still maintain more than 95% of the highest excitation efficiency between 488-526nm. The emission peak of CyOFP ...

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Abstract

The invention provides large stokes displacement fluorescent protein CyOFP and application of the fluorescent protein to microimaging and highly-sensitive living creature light-emitting imaging. The fluorescent protein is obtained through mNeptune site directed mutagenesis, the 1-230 positions of the amino acid sequence of the fluorescent protein sequentially correspond to 4-234 positions shown by the SEQ ID No: 2, or the fluorescent protein can be further obtained by conducting gene synthesizing on the fluorescent protein DNA segment and expression. The invention further provides a novel BRET system and fusion protein Antares obtained through system optimization. The CyOFP can be stimulated by blue light, and a quite high quantum yield is achived; the CyOFP and the enhanced green fluorescent protein (EGFP) are jointly stimulated by monochromatic light at the same time in a two-photon mode, and the resolution ratio of two-photon imaging is raised; a novel BRET system formed together with fluorescent protease NanoLuc greatly improves the depth and sensitivity of living creature imaging.

Description

technical field [0001] The invention belongs to the technical field of biomedical optics and molecular imaging, and specifically relates to a large Stokes shift fluorescent protein CyOFP and an application thereof. Background technique [0002] In the field of biomedical optics and molecular imaging, fluorescent proteins have important applications in the study of gene expression levels, protein interactions, protein conformations, and protein activities in living cells. However, when using fluorescent proteins for in vivo imaging, it is often difficult to achieve simultaneous multi-channel imaging. If this limitation can be overcome, the detection of multiple biological rapid response events can be realized, which will greatly promote the development of neuroscience. In single-photon imaging, multiple fluorescent proteins can be excited by light with different excitation wavelengths to achieve multi-channel simultaneous imaging. However, in multiphoton imaging, the cost of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/00C12N15/11C07K19/00A61K49/00
CPCC07K14/00A61K49/0045C07K2319/60C07K2319/61
Inventor 储军张楚秋王慧娜郭育奇
Owner SHENZHEN INST OF ADVANCED TECH
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