Fluorescent protein pair for highly sensitive FRET imaging and application thereof

Abstract

The invention provides a fluorescent protein pair for highly sensitive FRET imaging and application thereof. Specifically, a red fluorescent protein is provided. Compared with the amino acid sequenceof mRuby2, the amino acid sequence of the red fluorescent protein has the following mutation sites: N33R, M36E, T38V, K74A, G75D, M105T, C114E, H118N, Q120K, H159D, M160I, S171H, S173N, I192V, L202I,M209T, F210Y, H216V, F221Y, A222S and G223N. The invention also provides a green fluorescent protein with the amino acid sequence having the following mutation sites compared to the amino acid sequence of Clover: N149Y, G160S or G160C and A206K. The red fluorescent protein and the green fluorescent protein have good photostability and can be used for highly sensitive FRET imaging.

Description

[0001] The present invention is a divisional application with the filing date of December 31, 2015, the application number of 201511025610.8, and the title of the invention "a fluorescent protein pair that can be used for high-sensitivity FRET imaging and its application", and its entire content is incorporated into this application by reference invention. technical field [0002] The present invention relates to pairs of fluorescent proteins that can be used for highly sensitive FRET imaging and their applications. Background technique [0003] Fluorescence resonance energy transfer (FRET) is an energy transfer phenomenon between two fluorescent molecules that are very close to each other. When the emission spectrum of the donor fluorescent molecule overlaps with the absorption spectrum of the acceptor fluorescent molecule, and the distance between the two molecules is within 10nm, a non-radioactive energy transfer occurs, that is, the FRET phenomenon, so that the fluoresce...

AI Technical Summary

Problems solved by technology

However, this biosensor has many defects, which are mainly manifested in the following aspects: (1) The cyan and yellow fluorescent proteins used in ordinary biosensors have a low dynamic sensitivity range, so the imaging sensitivity is low, and it is difficult to detect intracellular Some transient and weak biochemical reactions are monitored

(2) The phototoxicity is high. When detecting living cells or samples, it will have a large negative impact on the normal metabolic reactions and molecular interactions of cells, resulting in large errors in experimental results. Cells may be in the long-term imaging process. die

(5) The fluorescent protein used in the sensor is sensitive to pH. When the pH changes slightly, the fluorescent protein may be inactivated and the fluorescence will be significantly weakened.

But the disadvantage is that Clover's photostability is weak, and it is easy to photobleach under continuous light irradiation.

The light intensity of mRuby2 is not high, so the use of this fluorescent protein FRET pair is limited (Lam, A.J.etal. Improving FRET dynamic range with bright green and red fluorescent proteins. Nat Methods9, 1005-1012(2012))

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