CN105504027B - Fluorescent protein pair for high-sensitivity FRET imaging and application thereof - Google Patents
Fluorescent protein pair for high-sensitivity FRET imaging and application thereof Download PDFInfo
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Abstract
Specifically, the invention provides a red fluorescent protein, and compared with the amino acid sequence of mRuby2, the amino acid sequence of the red fluorescent protein has the following mutation sites of N33R, M36E, T38V, K74A, G75D, M105T, C114E, H118N, Q120K, H159D, M160I, S171H, S173N, I192V, L I, M209T, F210Y, H216V, F221Y, A S22 2, and G223N.
Description
Technical Field
The invention relates to a fluorescent protein pair for high-sensitivity FRET imaging and application thereof.
Background
Fluorescence Resonance Energy Transfer (FRET) is an energy transfer phenomenon that occurs between two fluorescent molecules in close proximity. When the emission spectrum of the donor fluorescent molecule overlaps the absorption spectrum of the acceptor fluorescent molecule and the distance between the two molecules is within 10nm, a non-radioactive energy transfer, FRET, occurs, so that the fluorescence intensity of the donor is much lower than it would be if it were present alone (fluorescence quenching) while the fluorescence emitted by the acceptor is greatly enhanced (sensitized fluorescence). In an organism, two protein molecules are generally considered to have a direct interaction if the distance between the two protein molecules is within 10 nm. The fluorescence resonance energy transfer technology can monitor the activity of biomolecules in living cells and the mutual reaction between the biomolecules in real time, and provides an effective method with high time and spatial resolution for researching complex cell signal paths. With the development of green fluorescent protein application technology, FRET has become a powerful tool for detecting the nano-scale distance and the change of the nano-scale distance of biomacromolecules in living bodies, and has wide application in the aspects of biomacromolecule interaction analysis, cell physiological research, immunoassay and the like.
In the field of bio-optics and molecular imaging, scientists are constantly striving to find efficient gene-coded biosensors that can be used for optical imaging. These gene-encoded biosensors monitor the activity of biomolecules and the interaction between molecules in real time using fluorescence resonance energy transfer technology. The gene coding biosensor widely used at present is that cyan and yellow fluorescent proteins are fused, two colors of green and yellow fluorophores are matched, and the activity of biological molecules and the interaction between the molecules are monitored in real time through fluorescence resonance energy transfer. However, such biosensors have many drawbacks, mainly expressed in the following aspects: (1) the dynamic light sensing range of two fluorescent proteins, cyan and yellow, used in a common biosensor is low, so that the imaging sensitivity is low, and some transient and weak biochemical reactions in cells are difficult to monitor. (2) The phototoxicity is high, and when living cells or samples are detected, normal metabolic reactions and molecular interactions of the cells are greatly influenced, so that the experimental result has large errors, and the cells can die in the long-time imaging process. (3) Biosensor autofluorescence interference. When imaging in cells, the sensor inevitably excites cyan fluorescent protein and simultaneously excites some endogenous molecules in the cells, such as flavin, so that autofluorescence can occur, and therefore, the experimental result can be interfered and influenced to different degrees. (4) The cyan fluorescent protein appears photoactivated upon excitation of the yellow fluorescent protein. (5) The fluorescent protein used by the sensor is sensitive to pH, and when the pH is slightly changed, the fluorescent protein can be inactivated, and the fluorescence can be obviously weakened.
L am, A.J. and the like in 2012 developed a GFP-RFP pair, two fluorescent proteins are Clover and mruby2 respectively, the FRET pair of the fluorescent protein has improved reaction sensitivity compared with the common CFP-YFP FRET pair, reduces phototoxicity during imaging, has superior properties compared with other GFP-RFP pairs, and the scheme has also been successfully applied to Zn in living cells2+Aggregation and imaging of CaMKII α activity the inadequacies were the weak photostability of Clover and the susceptibility to photobleaching under continuous light irradiation.The light intensity of mRuby2 is also not high, so the use of the fluorescent protein FRET pair is limited (L am, a.j. et. improving FRET dynamic range with bright green and red fluorescent proteins. nat methods9,1005-1012 (2012)).
Disclosure of Invention
The invention mainly aims to find the efficient fluorescent protein of the gene coding biosensor which can be used for optical imaging.
According to the invention, amino acid site mutation is carried out on the basis of fluorescent proteins mRuby2 and mClover in the prior art by a site-directed mutagenesis technology to obtain mutated new red fluorescent protein and green fluorescent protein, so that the photophysical properties of the proteins are improved.
Specifically, in one aspect, the invention employs site-directed mutagenesis based on mRuby2 to obtain the novel red fluorescent protein of the invention. Compared with the amino acid sequence of mRuby2 (the amino acid sequence of mRuby2 can be seen in figure 1a), the amino acid sequence of the novel red fluorescent protein has the following mutation sites: M160I. M160I can make the mutant protein brighter than mRuby2, and a protein with M160I mutation based on mRuby2 has higher light intensity than unmutated mRuby 2.
According to a particular embodiment of the invention, the amino acid sequence of the novel red fluorescent protein of the invention has, compared to the amino acid sequence of mRuby2, the following mutation sites N33R, M36E, T38V, K74A, G75D, M105T, C114E, H118N, Q120K, H159D, S171H, S173N, I192V, L202I, M209T, F210Y, H216V, F221Y, A222S, G223N in combination with one or more of these mutation sites which allow the protein to mature or fold better.
(a) A protein having an amino acid sequence shown as SEQ ID No. 2;
(b) and (b) a protein derived from (a) by substituting, deleting or adding one or more amino acids in the amino acid sequence defined in (a) and having the same function as (a). Wherein the "same function" refers to a function with improved photophysical properties (e.g., improved brightness) compared to the mRuby2 protein.
According to a preferred embodiment of the present invention, the amino acid sequence of the novel red fluorescent protein of the present invention is shown in SEQ ID No.2 (wherein, the M160I mutation site corresponds to the 164 th position of the amino acid sequence of SEQ ID No.2, the N33R mutation site corresponds to the 37 th position of the amino acid sequence of SEQ ID No.2, … …, and so on), and the protein is named mRuby3 in the present invention. The sequence of the gene which encodes mRuby3 of the protein and is designed by the invention is shown in SEQ ID No. 1.
The excitation and emission spectra of mRuby3 peaked at about 558nm and 592nm, respectively, with a blue shift compared to mRuby 2. Extinction coefficient at peak 128mM-1cm-1The quantum yield is 0.45, so the light intensity of mRuby3 is 35% higher than mRuby2, so it is the brightest monomeric red fluorescent protein so far. In addition, mRuby3 has good light stability, and the half-life of mRuby3 under the irradiation of an arc lamp is 349 seconds, which is longer than 123 seconds of mRuby2 and 337 seconds of TagRFP-T. In terms of photobleaching kinetics, mRuby3 exhibited a single exponential relationship with a dissociation constant value of 4.8 and similar acid resistance compared to mRuby 2. mRuby3 is the red fluorescent protein with the brightest brightness and the best light stability so far
In another aspect, the invention also provides a fusion protein of mRuby3, for example, a fusion protein of mRuby3 with mClover, mClover3, meneongreen, or EGFP. Experiments prove that the fusion protein of mRuby3 can be accurately combined with an important subcellular target region in a mammalian cell line. mRuby3 expressed a signal intensity in mammalian cell lines that was at least 100% higher than the red fluorescent proteins mRuby2, fusion red and mCherry. mRuby3 is a more efficient fluorescence resonance energy transfer receptor than mRuby 2.
On the other hand, the invention also modifies the green fluorescent protein Clover to obtain the new green fluorescent protein which has improved brightness and light stability and can be used as an mRuby3 fluorescence resonance energy transfer efficient donor.
According to a specific embodiment of the present invention, the amino acid sequence of the novel green fluorescent protein provided by the present invention has the following mutation sites compared with the amino acid sequence of Clover: N149Y.
According to a preferred embodiment of the present invention, the amino acid sequence of the novel green fluorescent protein of the present invention further has the following mutation sites compared to the amino acid sequence of Clover: G160S or G160C;
according to a preferred embodiment of the present invention, the amino acid sequence of the novel green fluorescent protein of the present invention further has the following mutation sites compared to the amino acid sequence of Clover: a 206K.
According to a specific embodiment of the present invention, the present invention provides a novel green fluorescent protein which is a protein selected from the following (a) or (b):
(a) a protein having an amino acid sequence as shown in SEQ ID No.4, SEQ ID No.5 or SEQ ID No. 6;
(b) and (b) a protein derived from (a) by substituting, deleting or adding one or more amino acids in the amino acid sequence defined in (a) and having the same function as (a). Wherein "same function" refers to a function with improved photophysical properties (e.g., improved photostability) as compared to the Clover protein.
In a specific embodiment of the present invention, the amino acid sequence of the novel green fluorescent protein of the present invention has only the following mutation sites compared to the amino acid sequence of Clover: N149Y. The invention names the novel green fluorescent protein as clover1.5, and particularly, the amino acid sequence of the novel green fluorescent protein is shown in SEQ ID No.5 (wherein, the N149Y mutation site corresponds to the 150 th position of the amino acid sequence of the SEQ ID No. 5).
In a specific embodiment of the present invention, the amino acid sequence of the novel green fluorescent protein of the present invention has only the following two mutation sites compared to the amino acid sequence of Clover: N149Y and G160S. The novel green fluorescent protein is named dClover2, and specifically, the amino acid sequence of the novel green fluorescent protein is shown in SEQ ID No.6 (wherein, the N149Y mutation site corresponds to the 150 th position of the amino acid sequence of the SEQ ID No.6, and the G160S mutation site corresponds to the 161 th position of the amino acid sequence of the SEQ ID No. 6).
In a specific embodiment of the present invention, the amino acid sequence of the novel green fluorescent protein of the present invention has only the following three mutation sites compared to the amino acid sequence of Clover: N149Y, G160C and a 206K. The invention names the novel green fluorescent protein as mClover3, and specifically, the amino acid sequence of the novel green fluorescent protein is shown in SEQ ID No.4 (wherein, the N149Y mutation site corresponds to the 150 th site of the amino acid sequence of SEQ ID No.4, the G160C mutation site corresponds to the 161 th site of the amino acid sequence of SEQ ID No.4, and the A206K mutation site corresponds to the 207 th site of the amino acid sequence of SEQ ID No. 4), and the preferred gene sequence for coding the protein mClover3 designed by the invention is shown in SEQ ID No. 3.
The novel green fluorescent protein of the present invention has a half-life of dClover2 of 98 seconds in vivo under brightness normalization, a half-life of mClover3 of 80 seconds in vivo under brightness normalization, and Clover is only 50 seconds.
In another aspect, the invention also provides a fusion protein of the green fluorescent protein, for example, a fusion protein of mClover3 and mRuby 3. By systematically testing mCLOVER3 in mammalian cell lines, mCLOVER3 fusion protein was demonstrated to be able to accurately bind to important subcellular target regions in mammalian cell lines, mCLOVER3 and mNeonGreen had higher fluorescence resonance energy transfer efficiency than mEGFP, mCLOVER3-mRuby3 and mNeonGreen-mRuby3 were also more efficient than ClOVER-mRuby3, and mCLOVER3 and mNeonGreen were the most efficient donors of mRuby 3.
On the other hand, the invention also provides application of the red fluorescent protein as a fluorescence resonance energy transfer receptor. Specifically, the donor of the fluorescence resonance energy transfer can be selected from the following proteins: mEGFP, Envy, mNeonGreen, Clover or the novel green fluorescent protein of the invention.
The invention also provides application of the green fluorescent protein as a fluorescence resonance energy transfer donor. Specifically, the receptor of fluorescence resonance energy transfer is selected from the following proteins: mCherry, mKate2, fusion Red, mRuby2, or mRuby 3.
The invention also provides a protein pair for fluorescence resonance energy transfer imaging, which comprises:
the novel red fluorescent protein is used as a fluorescence resonance energy transfer receptor; and/or
The novel green fluorescent protein of the invention is used as a fluorescence resonance energy transfer donor.
The present invention also provides a gene-coded biosensor useful for optical imaging, the biosensor comprising:
the novel red fluorescent protein is used as a fluorescence resonance energy transfer receptor; and/or
The novel green fluorescent protein of the invention is used as a fluorescence resonance energy transfer donor.
Compared with the commonly used gene coding biosensor, the biosensor can effectively reduce imaging background interference signals in the fluorescence imaging process, remarkably improve the sensitivity of fluorescence imaging and enable the imaging to be clearer. The light stability of the sensor is greatly improved, and the imaging time is effectively prolonged, so that a certain reaction or phenomenon can be observed more easily for a long time. Due to the reduction of phototoxicity of the sensor, the influence on normal reaction and function of cells in the experimental process is reduced, so that the experimental error is reduced, and the experimental result is closer to the reality.
Specifically, in a specific embodiment of the present invention, Camui α sensor Camui α -CR3, mClover3 and mRuby3, Camui α sensor Camui α -C3R3, and meneon green and mRuby3, Camui α sensor Camui α -NR3, which are FRET pairs, were constructed with Clover and mRuby3, respectively, and the results of testing the new red and green fluorescent proteins in Camui α indicator for their ability to increase baseline FRET show that mClover3-mRuby3 and meneon green3-mRuby3 improve the reaction effect in Camui α.
In another aspect, the invention also provides a standard set of methods for assessing fluorescent proteins as independent markers and gene-encoded biosensors in mammalian cell lines by which the intensity of fluorescent signals generated by expression of fluorescent protein derivatives in mammalian cell lines is examined.
Compared with the widely used gene coding biosensor, the invention has 5 advantages: (1) the green and red fluorescent proteins are selected to be fused to replace the original cyan and yellow fluorescent proteins, and the fluorophores of the green and red colors are matched to increase the distance of the spectrum separation, reduce the interference and improve the sensitivity. (2) The light stability of the fluorescent protein is greatly improved, and the fluorescent protein mClover3 is improved by 60 percent compared with the previous generation Clover. Compared with the previous generation mRuby2, the mRuby3 is improved by 200%, so that the mRuby3 protein becomes the monomeric red fluorescent protein with the strongest light stability. (3) The brightness of the fluorescent protein is also greatly improved, for example, the mRuby3 is 35% brighter than the previous generation mRuby2, so that mRuby3 is the brightest red fluorescent protein so far. (4) High expression, and higher sensitivity for cell imaging than the biosensor commonly used at present after transfecting a mammalian cell line. (5) The mRuby3 is a good probe for detecting proteins under the conditions of delayed shooting imaging and limited light quanta, and comprises rapid delayed shooting imaging and single molecule imaging.
Drawings
FIG. 1a shows the sequence and alignment of the primary structures of mRuby3 and mRuby2 proteins.
FIG. 1b is a diagram of mutant comparative crystals of mRuby3 and mRuby 2.
Fig. 1c is an absorption (left) and emission (right) plot of the red fluorescent proteins mCherry, mKate2, fusion red, mRuby2, and mRuby 3.
FIG. 1d shows the fluorescence of the mRuby3, mRuby2 and mRuby2-M160I mutants after culture in a bacterial incubator.
FIG. 2a is a graphic image of the fluorescence image of a specific subcellular structure after expression of mRuby3 fusion protein in He L a cells, from left to right, mRuby3-7aa-actin (actin cytoskeleton), mRuby3-6aa-tubulin (microtubule), connexin43 (cell adhesion junction) -7aa-mRuby3, mRuby3-10aa-H2B (nucleosome), middle aa represents the unit of distance connecting two proteins, with a single amino acid distance of 1 aa.
Figure 2b shows a comparison of the brightness of the four monomeric red fluorescent proteins mCherry, fusion red, mRuby2 and mRuby3 in HEK293A and He L a cells.
FIG. 3a shows the sequence and alignment of the primary protein structures of mClover3, dCover 2 and Clover.
FIG. 3b is a crystal structure diagram of the mutation site of mClover3 compared to Clover.
FIG. 3c is a graph of the absorption (left) and emission (right) spectra of the green fluorescent proteins mEGFP, Envy, mNeonGreen, Clover and Clover 3.
FIG. 4a is a photograph of a fluorescent image of a specific subcellular structure after expression of mClover3 fusion protein in He L a cells, from left to right, mClover3-7aa-actin (actin cytoskeleton), mClover3-6aa-tubulin (microtubule), connexin43 (cell adhesion junction) -7aa-mClover3, mClover3-10aa-H2B (nucleosome), middle aa represents a unit of distance connecting two proteins, with a single amino acid distance of 1 aa.
FIG. 4b shows a comparison of the brightness of six monomeric green fluorescent proteins by expression of GFP-P2A-mCherry in HEK293A and He L a cells.
FIG. 4c is a graph comparing the efficiency of fluorescence resonance energy transfer of three GFP-mRuby3 proteins in HEK293A and He L a cells.
FIG. 5a shows the structure of a green/red FRET pair linked to Camui α.
Fig. 5b shows the donor/acceptor emission Ratio (RDA) of green/red He L a cells expressing Camui α in the absence of calcium ionophore stimulation.
In FIG. 5C, the left panel is a plot of the mean donor/acceptor emission Ratio (RDA) versus time and the right panel is a plot of the intensity ratio obtained from He L a cells in response to calcium ionophore stimulation.
Detailed Description
In order that the invention may be more clearly understood, it will now be further described with reference to the following examples and the accompanying drawings. The examples are for illustration only and do not limit the invention in any way. In the following experiments, the raw reagent materials are commercially available, and the experimental methods including site-directed mutagenesis, fusion protein, artificial liposome cell transfection and the like, which are well known in the art, and conventional conditions, or conditions suggested by the instrument manufacturer, are not specified.
The main techniques and experimental schemes used in the experiments of the invention include:
(1) distance model of fluorescence resonance energy transfer.
The formula of the established model is
Indicating the intensity of fluorescence at a certain wavelength.D(λex) Representing the extinction coefficient of the donor at the excitation wavelength. And E represents the fluorescence resonance energy transfer efficiency.
Is a quantum yield representing the donor. f. ofD(λ) represents the normalized emission of the donor at the wavelength λ.A(λex) Is an expression of the extinction coefficient of the acceptor at the excitation wavelength. And the internal fluorophore distance (r) is determined by Forster: (R)
) Formula E is 1/(1+ (r)6/r0 6) Calculating r0Is Foster: (
) A radius.
(2) Construction of recombinant plasmids
Plasmid construction includes Polymerase Chain Reaction (PCR), overlap extension polymerase chain reaction (overlap PCR), restriction enzyme digestion and In-Fusion ligation, transformation of DH5 α bacteria.
(3) He L a and HEK293A cell culture and transfection
Transferring the recombinant plasmid into He L a and HEK293A cells by a liposome transfection method, replacing the culture solution after 3-5 hours, digesting the cells after 24 hours of culture, subpackaging the cells into 8-pore plates, and culturing for 24 hours.
(4) Fluorescence microscopy of fusion proteins
For mClover3-mRuby3 fusion protein, after 24-72 hours of transfection into He L a cells, imaging by using FV1000 laser confocal microscope, for mClover3, excitation was performed at 488nm excitation light, light at 500-600nm was collected, for mRuby3, excitation was performed at 559nm, light at 570-670nm was collected, and the obtained image was processed by software ImageJ.
(5) In vitro characterization of fluorescent proteins
Since the N-terminus of the fluorescent protein was linked to six histidines, cobalt resin purification (HisPur CobaltResin) was used. And the absorbance, excitation spectrum and emission spectrum are measured by a multifunctional microplate reader SafireII. The extinction coefficient was determined by a denaturation method (for each protein sample, two identical solutions were prepared, proteins were dissolved in PBS, one of them measured the absorption peak, the other measured the absorption peak under the action of 1N NaOH, the extinction coefficient was calculated by the formula 'pre-denaturation/post-denaturation 44000'), and the quantum yield was referenced by Clover and mRuby2 (emission spectrum and absorption spectrum of the sample to be measured and the reference protein were measured respectively, then the emission spectrum area and the absorption value of the emission spectrum were calculated respectively, then the emission spectrum area was divided by the absorption value, then the above ratio of the sample to be measured was divided by the ratio of the reference protein, then multiplied by the quantum yield of the reference protein, and the quantum yield of the protein to be measured was obtained).
In an in vitro photobleaching experiment, purified protein was sampled using an inverted fluorescence microscope oil-scope, using a 40 × 0.90.90-NA UPlan S-Apo objective, using an X-Cite 120-Watt metal halide lamp at 100% neutral density through an excitation filter of 545/30nm (for the mRuby mutant) and an excitation filter of 485/30nm (for the Clover mutant), respectively, under continuous irradiation with a metal chloride light source, images were taken every 1 second (camera ORCA-ERCCD), again adjusted to produce a photon output rate of 1000 photons per second.
A complex gel column (Superdex 20030/100G L column) was used in the gel filtration chromatography experiments, the loading was 100. mu. L, the concentration was 10. mu.M, the elution fluidity was 0.5 ml/min, and the protein elution was monitored by absorption at 280 nm.
(6) Measurement of fundamental fluorescence resonance energy transfer
GFP-RFP fusion protein from C-end truncated av GFP derivatives or mNeonGreen through connecting sequences fused mRuby2 or mRuby3 aa 3-233, transfected into HEK293A and He L a cells, 2 days after transfection, cells were transferred to 96-well plates for fluorescence spectrum detection, emission spectrum range of 490-750nm, with 470nm excitation.
(7) Comparison of mutant lightness in mammalian cell lines
When comparing the green fluorescent protein and the mutant of mRuby in a mammalian cell line, mCherry and mTurquoise2 are taken as expression internal references, a liposome method is used for transfecting recombinant plasmids into HEK293A and He L a cells, the cells are transferred into a 96-well plate with a transparent bottom for detecting a fluorescence spectrum 2 days after transfection, and the setting parameters of different fluorescent proteins are as follows, mTurquoise2-434/5nm-474/5nm, mChery-587/20 nm-610/5nm, GFP-430/20 nm-480-650 nm, RFP-550/10 nm-570-670 nm, and the relative brightness is obtained by dividing the emission light intensity of the integrated green fluorescent protein or mRuby mutant by the emission light intensity of mCherry or mTurquoise 2.
(8) Camui α sensor improvements and features
To construct the Camui α -CR mutant, an N-terminal NheI cleavage site and a C-terminal extension-encoded linker (between GFP and CaMKII α) were used to amplify C-terminal truncated avGFP (without the ` GITHGMDE L YK ` sequence) or mNeonGreen (without the ` GMDE L YK ` sequence). after successful amplification by PCR of the CaMKII α region at either end, the desired fragment was also amplified by PCR for either mRuby2 or mRuby3, simultaneously introducing an N-terminal extension linker in the CaMKII α region, an ApaI cleavage site at the C-terminal, and the insert fragment was cloned into the vector pcDNA3.1 by overlap extension polymerase chain reaction.
Cells were observed microscopically 2 days after transfection with recombinant plasmid, observed using a cold ORCA-ER CCD camera of an inverted fluorescence microscope 200M and a 40 × 1.2.2-NA C-high achromatic mirror water immersion module, using software Micro-manager1.4 with the specific parameters 17-inch 2.5-GHz Core 2Duo MacBook Pro running Mac OS 10.6.8 continuous FRET and donor emission spectral imaging was obtained using filters with green fluorescent protein excitation HQ470/30nm and emission 505AE L P nm, FRET excitation HQ470/30nm and emission BA575IF nm.
(9) Ratio type image analysis
Measurement of FRET was quantified by software ImageJ. The original file was a 16-bit TIFF file, fields were selected by random methods, transfected fluorescent cells were positive and untransfected cells were used as background. The emission ratio is the value obtained by dividing the background-removed emission intensity of the donor by the background-removed FRET intensity. An intensity modulation display was generated by the receptor channel using a full spectrum look-up table (blue minimum and red maximum).
(10) Statistical method
The difference in brightness measurements in cells was determined using ANOVA and Dunnite assay A T-test was used to determine whether there was a statistical difference in the change in peak emission ratio for the Camui α mutant.
Example 1: fluorescent protein pairs useful for high sensitivity FRET imaging
1 Properties and imaging Effect of the New Red fluorescent protein mRuby3
1.1 Structure and photophysical Properties of mRuby3
The new red fluorescent protein mRuby3 is obtained by carrying out site-directed mutagenesis on the basis of mRuby2 by adopting a site-directed mutagenesis technology. The amino acid sequence of the mRuby3 is shown in SEQ ID No.2, and the gene sequence of the mRuby3 which is designed and preferably encodes the protein is shown in SEQ ID No.1 (the DNA sequence of mRuby3 can be obtained by a gene synthesis method based on PCR polymerization, namely, a plurality of PCR primers are designed, each two primers are overlapped by 18-25 bp, and then all the primers are polymerized by an overlap PCR method, so that a complete gene is obtained).
The mRuby3 has 21 substitutions compared with the mRuby of the previous generation, specifically N33, M36, T38, K74, G75, M105, C114, H118, Q120, H159, M160, S171, S173, I192, 202I, M209, F210, H216, F221, A222, G223, as seen in FIG. 1a, while the crystal structure of mRuby (PDB database ID 3U 0) as seen in FIG. 1b, the amino acids forming the chromophore have been marked with black boxes, mutations located on the outer wall of the protein are marked with blue, including N33, T38, M105, C114, H118, Q120, H159, S171, S173, 202I, F210, H216, mutations located on the inner wall are marked with green, including M160, mutations located on the loop are marked with orange, including M36, K74, G75, I221, G209, G223, M209, G223.
The absorption profiles of the red fluorescent proteins mCherry, mKate2, fusion red, mRuby2, and mRuby3 can be seen in the left panel of fig. 1c, and the emission profiles of the respective proteins can be seen in the right panel of fig. 1 c. It can be seen that the excitation and emission spectra of mRuby3 have peaks at 558nm and 592nm, respectively, with a blue shift compared to mRuby 2. Extinction coefficient at peak 128mM-1cm-1The quantum yield is 0.45 (see table 1), so the light intensity of mRuby3 is 35% higher than that of mRuby2, so it is the brightest monomeric red fluorescent protein so far. In addition, mRuby3 has good light stability, and the half-life of mRuby3 under the irradiation of an arc lamp is 349 seconds, which is longer than 123 seconds of mRuby2 and 337 seconds of TagRFP-T. In terms of photobleaching kinetics, mRuby3 exhibited a single exponential relationship with a dissociation constant value of 4.8 and similar acid resistance compared to mRuby 2. Therefore mRuby3 is the red fluorescent protein with the brightest brightness and the best light stability so far.
Table 1: photophysical properties of monomeric green and red fluorescent proteins
And (4) surface note: b is the wavelength at which the peak of the excitation light is located. c is the wavelength at which the emitted light peaks. d is the maximum extinction coefficient. e is the fluorescence quantum yield. f is the relative value of the protein brightness. g is the dissociation constant. h is the photostability of the protein, which represents the time taken for photobleaching from 1000 photons per second to 500 photons per molecule in seconds under arc lamp illumination.
In each mutation site of mRuby3 compared to mRuby2, M160I made the protein brighter, but protein maturation or folding became worse; while other sites allow for better protein maturation or folding. In the invention, site-directed mutagenesis is also carried out on the basis of mRuby2 to obtain a mutant which only has an M160I mutation site compared with mRuby2 and is named mRuby2-M160I in the invention. Panel (1) in FIG. 1d shows the fluorescence profiles of mRuby2 (right) and mRuby2-M160I (left) mutants incubated overnight in a bacterial incubator for 24h, and panel (2) in FIG. 1d shows the fluorescence profiles of mRuby2 (right) and mRuby2-M160I (left) mutants incubated in a bacterial incubator for 48 h. As can be seen, mRuby2-M160I was slightly darker than mRuby2 after 24h culture due to slower maturation or folding, but eventually brighter than mRuby2 as the culture time increased. Panel (3) in FIG. 1d is a fluorescence plot of mRuby3 and mRuby2 after 24h overnight incubation in a bacterial incubator (mRuby 3 on the left and mRuby2 on the right in the figure).
1.2mRuby3 imaging in mammalian cell lines
FIG. 2a is a photograph of an image of fluorescence imaging of a specific subcellular structure after expression of mRuby3 fusion protein in He L a cells from left to right: mRuby3-7aa-actin (actin cytoskeleton), mRuby3-6aa-tubulin (microtubule), connexin43 (cell adhesion junction) -7aa-mRuby3, mRuby3-10aa-H2B (nucleosome). intermediate aa represents a unit for connecting the distance between two proteins, and mRuby3 fusion protein (PCR amplification to obtain mRuby3 sequence) was demonstrated at a single amino acid distance of 1 aa. this was then ligated into GFP-C1 vector with BglII double digestion to construct pmRuby 3-C733, then separately amplified with subcellular localization sequences, then inserted into the EcoRI fusion protein in pmM 73784 vector of mammalian cells to construct an accurate fusion of target subcellular structure with EcoRI 4642.
FIG. 2b shows the brightness comparison of the four monomeric red fluorescent proteins mCherry, fusion Red, mRuby2 and mRuby3 in HEK293A and He L a cells, and the result shows that mRuby3 expresses the strongest signal intensity in HEK293A and He L a cells, which is at least 100% higher than that of the red fluorescent proteins mRuby2, fusion Red and mCherry (FIG. 2 b).
It can thus be seen that mRuby3 is theoretically an excellent acceptor for FRET. Since mRuby2 has been shown to be a highly efficient fluorescence resonance energy transfer receptor. Therefore comparing mRuby3 with mRuby2, with the same green fluorescent protein Clover as the donor, mRuby3 was found to be more efficient than mRuby 2.
2 Properties and imaging Effect of the New Green fluorescent protein mCrover 3
2.1 Structure and photophysical Properties of mClover3
In the invention, the green fluorescent protein Clover is also transformed, and the protein which has improved brightness and light stability and can be used as an mRuby3 fluorescence resonance energy transfer efficient donor is obtained. Obtaining a Clover mutant by a Random mutation technology (which is carried out by adopting a GeneMorph II Random Mutagenesis kit of Clontech company to ensure that 1-3 bp mutation/1000 bp), and then screening the mutant with strong light stability by the irradiation of a blue light-emitting diode. Screening gave Clovert 1.5 (asparagine to tyrosine at position 149) and dClover2 (aspartic acid to tyrosine at position 149 and glycine to serine at position 160). In vivo, the half-life of light stability at brightness normalization was 98 seconds for dClover2, while Clover was only 50 seconds. Extinction coefficient 123mM–1cm–1，Also compared to 111mM of Clover–1cm–1Is improved. The relative quantum yield was 0.8 slightly higher than 0.76 of Clover. On the basis of dCover 2After the alanine at the position 206 is changed into the lysine and the serine at the position 160 is changed into the cysteine, the monomeric fluorescent protein mClover3 is obtained. The amino acid sequence of mClover3 is shown in SEQ ID No.4, and the gene sequence of mClover3 which is designed and preferably encodes the protein is shown in SEQ ID No.3 (the DNA sequence of mClover3 can be obtained by a gene synthesis method).
FIG. 3a shows the sequence and alignment of the primary protein structures of mClover3, dCover 2 and Clover. The amino acids forming the chromophore have been marked with black boxes. Three mutations located on the protein wall (N149, G160, A206) are marked with orange color. Figure 3b shows the crystal structure of mClover3 at the mutation site compared to Clover. Figure 3c shows the absorption spectra (left panel) and emission spectra (right panel) of the green fluorescent proteins mcegfp, Envy, meneon green, Clover and Clover3, and it can be seen that mClover3 possesses a similar spectrum compared to Clover and is also comparable in brightness. Further mClover3 had a half-life of 80 seconds for light stability at brightness normalization when in vivo.
2.2mClover3 in mammalian cell lines
Figure 4a shows a plot of fluorescence images of specific subcellular structures after expression of mClover3 fusion protein in He L a cells, from left to right, mClover3-7aa-actin (actin cytoskeleton), mClover3-6aa-tubulin (microtubule), connexin43 (cell adhesion junction) -7aa-mClover3, mClover3-10aa-H2B (nucleosome), the middle aa representing the unit of distance between the two proteins, 1 aa apart by a single amino acid distance, demonstrating that the fusion protein of mRuby3 can accurately bind to important subcellular target regions in mammalian cell lines, then comparing the fluorescent signals generated by rumby and mRuby3 in mammalian cells with the green fluorescent proteins of other monomers, such as the fluorescent signals generated by the mcegfp, sfGFP and envegfy are now the most widely used fluorescent proteins, compared to the green fluorescent proteins of other monomers, but the fluorescent signals are considered to have a similar effect in vitro as the fluorescent spectra of the fluorescent protein of mClover3, but the fluorescent protein of the three types of mClover GFP, which are considered to have a similar effect in vitro as the fluorescence spectrum of the fluorescent protein of the mClover 3.
The brightness of six monomeric green fluorescent proteins was compared by expression of GFP-P2A-mCherry in HEK293A and He L a cells and as a result, see figure 4 b. the mean fluorescence intensity of cells transfected with meneon green was found to be brightest in HEK293A cells, followed by Clover or mClover3, again Envy, and the weakest was sfGFP. meneon green and mClover3, mClover3 and EGFP or sfGFP with statistical significance (P < 0.05). in He L a cells, nemongreen, Clover or mClover3 all showed strong fluorescence after expression, with little difference in brightness from each other, while Envy's brightness was second, followed by EGFP, and finally sfGFP. mClover3, egfvy, P and sfGFP.
In order to find the most suitable monomeric green fluorescent protein which can be used as the optimal donor of the fluorescent resonance energy transfer of the mRuby3, the monomeric green fluorescent proteins mChlorer, mChlorer 3, mNeonGreen and EGFP to be detected are made into fusion proteins with mRuby3, and then the fluorescent resonance energy transfer efficiency of various fusion proteins is detected in mammalian cells, and the result is shown in FIG. 4c, which shows that the emission light spectrum (red line) obtained by the experiment is matched with the linearly fitted emission light spectrum (black line), and the mChlorer 3 and mNeonGreen have higher fluorescent resonance energy transfer efficiency than mEGFP. mClover3-mRuby3 and mNeonGreen-mRuby3 were also more efficient than Clover-mRuby3 (FIG. 4 c). It can be seen that mClover3 and meneongreen are the most effective donors for mRuby 3.
3Camui α sensor
First, Camui α -CR3 with Clover and mRuby3 as FRET pairs, Camui α -C3R3 with mClover3 and mRuby3 as FRET pairs, and Camui α -NR3 with mNeonGreen and mRuby3 as FRET pairs were constructed, respectively.
FIG. 5a shows the structure of a green/red FRET pair linked to Camui α experiments demonstrate that when the activity of CaMKII α is detected, Clover3-mRuby3 improves the sensitivity of fluorescence resonance energy transfer in Camui α experiments in which a fluorescent protein is fused to the terminus of a CaMKII α polypeptide to form a FRET pair consistent with structural analysis, Camui α has high FRET capability in the inactive state and low FRET capability in the active state (FIG. 5 a).
Fig. 5b shows the donor/acceptor emission Ratio (RDA) of green/red He L a cells expressing Camui α without calcium ionophore stimulation, shows that Camui α -CR3 containing mRuby3 has a higher FRET baseline than Camui α -CR containing mRuby2 (fig. 5b), mere replacement of Clover with mClover3 or meneongreen does not significantly change the basal FRET levels (fig. 5b) data are presented as mean ± standard deviation.
Finally, it was examined whether the effect of Camui α itself was improved by the presence of a new FRET pair, comparing the response of Camui L-CR, -CR3, -C3R3 and-NR 3 in He L a cells under stimulation by calcium ionophore, the left panel of fig. 5C is a graph of the variation of the mean donor/acceptor emission Ratio (RDA) over time, the right panel is a graph of the intensity ratio resulting from the reaction of He L a cells under stimulation by calcium ionophore, the variation of the emission ratio of each cell is shown by grey lines in the graph, the mean is shown by black lines, the data is shown as mean ± standard deviation, the ratio of Camui L-CR to Camui L-C3R 3 and Camui L-NR 3 at the excitation peak, the average response of Camui α -CR3 is similar to Camui α -CR, approximately 45% (fig. 5) if Camui 8272-CR is improved significantly by Camui 3-NR3, the increase of the emission ratio of Camui 3-CR 3-NR3 in the range 3-NR 3-3, the increase of the emission ratio of Camui 3-CR, while the increase of the emission ratio of Camui 593-NR 3-C-3-C-3-C-b-3-C-b-3-C-3-b-C-b0Values and FRET baseline, but a significant increase in sensitivity to Camui α if mNeonGreen or mClover3 had two position mutations, 149 from N to Y and 206 from A to K, promoted the transition of Camui α -C3R3 and Camui α -NR3 to the activated state.
Claims (10)
1. A red fluorescent protein which is a protein selected from the following (a):
(a) the protein of the amino acid sequence shown as SEQ ID No. 2.
2. A polynucleotide encoding the red fluorescent protein of claim 1.
3. The polynucleotide of claim 2, wherein the polynucleotide has the sequence shown in SEQ ID No. 1.
4. A fusion protein comprising the red fluorescent protein of claim 1.
5. The use of the red fluorescent protein of claim 1 as a fluorescence resonance energy transfer receptor.
6. Use according to claim 5, wherein the donor of fluorescence resonance energy transfer is selected from the following proteins: mEGFP, Envy, mNeonGreen, Clover or the green fluorescent protein Clover1.5, dClover2 or mClover 3;
wherein the amino acid sequence of the green fluorescent protein Clover1.5 is shown as SEQ ID No. 5;
the amino acid sequence of the green fluorescent protein dClover2 is shown in SEQ ID No. 6;
the amino acid sequence of the green fluorescent protein mClover3 is shown in SEQ ID No. 4.
7. A pair of proteins useful for fluorescence resonance energy transfer imaging, comprising: the red fluorescent protein of claim 1, which acts as a fluorescence resonance energy transfer receptor.
8. The pair of proteins useful for fluorescence resonance energy transfer imaging according to claim 7, further comprising:
green fluorescent protein clover1.5, dClover2 or mClover3 as fluorescence resonance energy transfer receptor;
wherein the amino acid sequence of the green fluorescent protein Clover1.5 is shown as SEQ ID No. 5;
the amino acid sequence of the green fluorescent protein dClover2 is shown in SEQ ID No. 6;
the amino acid sequence of the green fluorescent protein mClover3 is shown in SEQ ID No. 4.
9. A gene-encoded biosensor useful for optical imaging, the biosensor comprising: the red fluorescent protein of claim 1 as a fluorescence resonance energy transfer receptor.
10. The genetically encoded biosensor useful for optical imaging according to claim 9, further comprising:
green fluorescent protein clover1.5, dClover2 or mClover3 as fluorescence resonance energy transfer receptor;
wherein the amino acid sequence of the green fluorescent protein Clover1.5 is shown as SEQ ID No. 5;
the amino acid sequence of the green fluorescent protein dClover2 is shown in SEQ ID No. 6;
the amino acid sequence of the green fluorescent protein mClover3 is shown in SEQ ID No. 4.
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