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Red fluorescence protein, fusion protein, separated nucleic acid, carrier and application

A red fluorescent protein, fluorescent protein technology, applied in the application, fusion polypeptide, fluorescence/phosphorescence, etc., can solve the problems of fast photobleaching, low protein maturation rate, unsatisfactory FRET effect, etc., achieve high FRET effect, broad application prospects Effect

Active Publication Date: 2017-05-10
SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, some donor-receptor pairs that can replace cyan and yellow fluorescent proteins have been reported in the literature, such as donor fluorescent protein: green fluorescent protein (GFP), yellow fluorescent protein; and acceptor fluorescent protein: orange Orange fluorescent protein (OFP), red fluorescent protein (RFP), also due to defects such as low protein maturation rate and fast photobleaching, the FRET effect is not ideal

Method used

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  • Red fluorescence protein, fusion protein, separated nucleic acid, carrier and application
  • Red fluorescence protein, fusion protein, separated nucleic acid, carrier and application

Examples

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Embodiment 1

[0059] The red fluorescent protein provided in this example has an amino acid sequence as shown in SEQ ID NO: 4, which has the following amino acid mutation sites relative to the fluorescent protein mRuby3 (SEQ ID NO: 3): N8E, R10P, E114V, V116I, T177E and R179K.

[0060] Among them, asparagine at position 8 is mutated to glutamic acid (N8E), the amino acid structure becomes larger, and the carboxyl group on the side chain of glutamic acid is more likely to form a hydrogen bond with the amino group of asparagine at position 33; Arginine at position 10 is mutated to proline (R10P), and glutamic acid at position 114 is mutated to valine (E114V), favoring nonpolar proline at position 10 and nonpolar proline at position 114 Valine forms a hydrophobic bond; the 116th valine is mutated to isoleucine (V116I), which makes the amino acid side chain longer, and the 114th glutamic acid is mutated to valine, which is beneficial to the 116th Isoleucine with a longer side chain forms a hyd...

Embodiment 2

[0065] On the basis of the red fluorescent protein (SEQ ID NO: 4) in Example 1, further site-directed mutations were carried out to improve its brightness, extinction coefficient, quantum yield and other characteristics.

[0066] The red fluorescent protein provided by this embodiment is named as mRuby4, and its amino acid sequence is shown in SEQ ID NO: 1. Compared with fluorescent protein mRuby3 (its amino acid sequence is shown in SEQ ID NO: 3), it not only has the following amino acid mutation sites: N8E, R10P, E114V, V116I, T177E and R179K; also have the following amino acid mutation sites: E16T, S18T, E111Q, V124E and N173S. The amino acid sequence alignment results of mRuby4 and mRuby3 are as follows figure 1 shown.

[0067] The red fluorescent protein (SEQ ID NO: 1) provided in this example not only has a high maturity rate, but also has high extinction coefficient, brightness and other characteristics.

[0068] The red fluorescent protein provided in this example ca...

Embodiment 3

[0070] This example provides a method for obtaining the red fluorescent protein (SEQ ID NO: 1) in Example 2. Of course, the method for obtaining the red fluorescent protein provided in Example 1 can also refer to this example. It should be noted that the method for obtaining the red fluorescent protein of the present invention is not limited to the method provided in this example. In other embodiments, the red fluorescent protein of the present invention obtained by other methods also belongs to the protection of the present invention. scope.

[0071] The method for obtaining red fluorescent protein (SEQ ID NO: 1) provided in this example is as follows.

[0072] (1) Design the corresponding nucleic acid sequence according to the amino sequence of the red fluorescent protein (SEQ ID NO:1) provided in Example 2, and the nucleic acid sequence encodes the red fluorescent protein (SEQ ID NO:1).

[0073] In this embodiment, the nucleotide sequence of the nucleic acid sequence encod...

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Abstract

The invention discloses a red fluorescence protein, a fusion protein, a separated nucleic acid, a carrier and an application, and belongs to the technical field of the biomedical optics and molecular imaging. Compared with the fluorescent protein mRuby3, the provided red fluorescence protein comprises the following amino acid mutation sites: combinations of one or more of N8E, R10P, E114V, V116I, T177E and R179K. The provided red fluorescence protein has photophysical properties, such as higher brightness, maturing rate and extinction coefficient, can be used for protein marking and imaging, or used as fluorescence resonance energy transfer acceptor or donor for application. The red fluorescence protein has better fluorescence resonance energy transfer efficiency.

Description

technical field [0001] The invention relates to the technical field of biomedical optics and molecular imaging, in particular to a red fluorescent protein, fusion protein, isolated nucleic acid, carrier and application. Background technique [0002] Fluorescence resonance energy transfer (FRET) is the non-radiative energy transfer of a donor fluorophore in its excited state, which transfers energy to an adjacent acceptor fluorophore, thus causing the acceptor fluorophore to emit fluorescence. Because FRET is highly sensitive to the distance between the donor and acceptor dipoles between 1-10nm, it is often used as an efficient optical molecular ruler and is widely used to monitor biochemical reactions that can produce changes in the distance between molecules, such as the study of biological macromolecules Interaction, immunoassay, nucleic acid detection, etc. Compared with other biochemical monitoring methods such as yeast two-hybrid, immunoprecipitation, etc., the advanta...

Claims

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Application Information

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IPC IPC(8): C07K14/435C07K19/00C12N15/12G01N21/64
CPCC07K14/43595C07K2319/00G01N21/6428
Inventor 储军张书张楚秋郭育奇刘丰
Owner SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI
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