CN105504027A - Fluorescent protein for high-sensitivity FRET imaging and application thereof - Google Patents
Fluorescent protein for high-sensitivity FRET imaging and application thereof Download PDFInfo
- Publication number
- CN105504027A CN105504027A CN201511025610.8A CN201511025610A CN105504027A CN 105504027 A CN105504027 A CN 105504027A CN 201511025610 A CN201511025610 A CN 201511025610A CN 105504027 A CN105504027 A CN 105504027A
- Authority
- CN
- China
- Prior art keywords
- fluorescent protein
- aminoacid sequence
- fret
- mruby3
- albumen
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Analytical Chemistry (AREA)
- General Engineering & Computer Science (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention provides a fluorescent protein for high-sensitivity FRET imaging and an application thereof, and particularly provides a red fluorescent protein. Compared with the amino acid sequence of 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 further provides a green fluorescent protein. Compared with the amino acid sequence of Clover, the amino acid sequence of the green fluorescent protein has the following mutation sites: N149Y, G160S or G160C and A206K. The red fluorescent protein and the green fluorescent protein have excellent photo-stability, and can be used in high-sensitivity FRET imaging.
Description
Technical field
The invention relates to the fluorescin that can be used for highly sensitive FRET imaging to and application.
Background technology
FRET (fluorescence resonance energy transfer) (fluorescenceresonanceenergytransfer, FRET) is a kind of energy transfer phenomenon apart from producing between two very near fluorescence molecules.When the emmission spectrum of donor fluorescent molecule is overlapping with the absorption spectrum of acceptor fluorescence molecule, and when the distance of two molecules is within 10nm scope, a kind of inactive energy trasfer will be there is, i.e. FRET phenomenon, many (quenching of fluorescence) that will be low when making its Individual existence of the fluorescence intensity ratio of donor, and the fluorescence of acceptor emission strengthens (sensitized fluorescence) greatly.And in vivo, if the distance of two protein molecules is within 10nm, it is generally acknowledged that these two protein molecules exist direct interaction.FRET (fluorescence resonance energy transfer) technology can monitor reacting to each other between the activity of biomolecules in viable cell and molecule in real time, for research complex cell signal path provides the effective ways of high Time and place resolving power.Along with the development of green fluorescent protein utilisation technology, FRET has become the powerful detecting biomacromolecule nano level Distance geometry nano level distance change in live body, has a wide range of applications in interaction of biomacromolecules analysis, cell physiological research, immunoassay etc.
In Photobiology and molecular image technical field, scientists is striving to find the genes encoding biosensor that can be used for optical imagery efficiently always.Reacting to each other between the activity of the monitoring biomolecules that the biosensor of these genes encodings utilizes FRET (fluorescence resonance energy transfer) technology real-time and molecule.The genes encoding biosensor generally used now is merged mutually with yellow fluorescence protein cyan, arranges in pairs or groups mutually, monitor the interaction between the activity of biomolecules and molecule in real time by FRET (fluorescence resonance energy transfer) with the fluorophore of blue or green and yellow two kinds of colors.But this biosensor has many defects, main manifestations is in the following areas: the dynamic sensitive volume of the cyan used in (1) common biosensor and yellow two kinds of fluorescins is lower, so cause image sensitivity low, be difficult to monitor more intracellular instantaneous and faint biochemical reactions.(2) phototoxicity is large, when detecting the cell or sample of living, producing larger negative impact to the normal metabolic reaction of cell and interaction of molecules, causing experimental result to occur larger error, and cell may be dead in long-time imaging process.(3) biosensor autofluorescence interference.In cell during imaging, due to sensor itself inevitably excite cyan fluorescent protein while can some endogenous molecules, such as flavin in activated cell, there will be autofluorescence, so interference in various degree and impact also can be caused on experimental result.(4) when exciting yellow fluorescence protein there is photoactivation in cyan fluorescent protein.(5) fluorescin that uses of sensor is responsive to pH, and when slight variations occurs pH, fluorescin may inactivation, and fluorescence can obviously weaken.
The people such as Lam, A.J. in 2012 develop a GFP-RFP couple, and two kinds of fluorescins are respectively Clover and mRuby2.This fluorescin FRET pair and common CFP-YFPFRET to compared with improve the susceptibility of reaction, phototoxicity when reducing imaging, with other GFP-RFP to compared with character superior, and the program has also been used successfully to Zn in viable cell
2+the imaging of gathering and CaMKII alpha active.But weak point is the light of Clover to be stablized weak, easy photobleaching under light Continuous irradiation.And the light intensity of mRuby2 is not high yet, so limit the right use (Lam of this fluorescin FRET, A.J.etal.ImprovingFRETdynamicrangewithbrightgreenandredf luorescentproteins.NatMethods9,1005-1012 (2012)).The full content of the document to be incorporated in the present invention as a reference at this.
Summary of the invention
The present invention is mainly in order to find the fluorescin of the genes encoding biosensor that can be used for optical imagery efficiently.
The present invention, by site-directed mutagenesis technique, amino acid sites sudden change is carried out on the basis of prior art fluorescin mRuby2 and mClover, and obtains new red fluorescent protein and the green fluorescent protein of sudden change, achieves the raising of opalescence physical performance.
Specifically, on the one hand, the present invention adopts site-directed mutagenesis technique on mRuby2 basis, carry out site-directed mutagenesis and obtains new red fluorescent protein of the present invention.The aminoacid sequence of the aminoacid sequence of this new red fluorescent protein of the present invention and mRuby2 (aminoacid sequence of mRuby2 can see Fig. 1 a) compared with, there is following mutational site: M160I.It is brighter that M160I can allow mutain compare mRuby2, and the albumen that mRuby2 basis has M160I sudden change has the light intensity higher than the mRuby2 do not suddenlyd change.
According to specific embodiment of the invention scheme, the aminoacid sequence of new red fluorescent protein of the present invention, compared with the aminoacid sequence of mRuby2, also has following mutational site: N33R, M36E, T38V, K74A, G75D, M105T, C114E, H118N, Q120K, H159D, S171H, S173N, I192V, L202I, M209T, F210Y, H216V, one or more combination in F221Y, A222S, G223N.These mutational sites can allow protein maturation or folding to improve.According to specific embodiment of the invention scheme, red fluorescent protein of the present invention, it is for being selected from the albumen of following (a) or (b):
A () has the albumen of aminoacid sequence as shown in SEQIDNo.2;
Through replacing, lacking or add one or several amino acid and with (a), there is the albumen derivative by (a) of identical function in b aminoacid sequence that () limits at (a).Wherein, described " identical function " refers to the function improving (such as brightness raising) compared to mRuby2 opalescence physical performance.
According to of the present invention one preferred specific embodiments, the aminoacid sequence of new red fluorescent protein of the present invention see shown in SEQIDNo.2 (wherein, the 164th of the corresponding SEQIDNo.2 aminoacid sequence in described M160I mutational site, the 37th of the corresponding SEQIDNo.2 aminoacid sequence in described N33R mutational site, the like), name this albumen to be mRuby3 in the present invention.The gene order of this albumen of preferred coding mRuby3 of the present invention's design is see shown in SEQIDNo.1.
At about 558nm and 592nm place, there is blue shift compared with mRuby2 respectively in the excitation spectrum of mRuby3 and the peak value of emmission spectrum.Optical extinction coefficient at peak value place is 128mM
-1cm
-1, quantum yield is 0.45, so the light strength ratio mRuby2 of mRuby3 exceeds 35%, so it is monomer red fluorescent protein the brightest so far.In addition, the light stability of mRuby3 is fine, and under the irradiation of Jupiter, the transformation period of mRuby3 is 349 seconds, is longer than 123 seconds of mRuby2 and 337 seconds of TagRFP-T.In photobleaching kinetics, mRuby3 shows single index relation, and its dissociation constant value is 4.8, and acid resistance is similar compared with mRuby2.MRuby3 is that the brightest and light of brightness so far stablizes best red fluorescent protein
On the other hand, present invention also offers the fusion rotein of mRuby3, such as, the fusion rotein of described mRuby3 and mClover, mClover3, mNeonGreen or EGFP.The fusion rotein that the present invention demonstrates mRuby3 by experiment can combine with the important subcellular object region in mammal cell line accurately.The strength of signal that mRuby3 is expressed in mammal cell line, exceeds at least 100% than red fluorescent protein mRuby2, FusionRed and mCherry.MRuby3 is a kind of FRET (fluorescence resonance energy transfer) acceptor more efficient than mRuby2.
On the other hand, equally also green fluorescent protein Clover is transformed in the present invention, obtain luminance brightness and light stability and to promote to some extent and can as the new green fluorescent protein of the efficient donor of mRuby3 FRET (fluorescence resonance energy transfer).
According to specific embodiment of the invention scheme, the aminoacid sequence of new green fluorescent protein provided by the invention, compared with the aminoacid sequence of Clover, has following mutational site: N149Y.
According to preferred specific embodiments of the present invention, the aminoacid sequence of new green fluorescent protein of the present invention, compared with the aminoacid sequence of Clover, also has following mutational site: G160S or G160C;
According to preferred specific embodiments of the present invention, the aminoacid sequence of new green fluorescent protein of the present invention, compared with the aminoacid sequence of Clover, also has following mutational site: A206K.
According to specific embodiment of the invention scheme, new green fluorescent protein provided by the invention, it is for being selected from the albumen of following (a) or (b):
A () has the albumen of aminoacid sequence as shown in SEQIDNo.4, SEQIDNo.5 or SEQIDNo.6;
Through replacing, lacking or add one or several amino acid and with (a), there is the albumen derivative by (a) of identical function in b aminoacid sequence that () limits at (a).Wherein, described " identical function " refers to the function improving (such as light stability raising) compared to Clover opalescence physical performance.
In a specific embodiments of the present invention, new green fluorescent protein of the present invention, its aminoacid sequence, compared with the aminoacid sequence of Clover, only has following mutational site: N149Y.The green fluorescent protein that the present invention names this new is Clover1.5, and particularly, its aminoacid sequence is see (the 150th of the corresponding SEQIDNo.5 aminoacid sequence in wherein, described N149Y mutational site) shown in SEQIDNo.5.
In a specific embodiments of the present invention, new green fluorescent protein of the present invention, its aminoacid sequence, compared with the aminoacid sequence of Clover, only has following two mutational site: N149Y and G160S.The green fluorescent protein that the present invention names this new is dClover2, particularly, its aminoacid sequence see shown in SEQIDNo.6 (wherein, the 150th of the corresponding SEQIDNo.6 aminoacid sequence in described N149Y mutational site, the 161st of the corresponding SEQIDNo.6 aminoacid sequence in described G160S mutational site).
In a specific embodiments of the present invention, new green fluorescent protein of the present invention, its aminoacid sequence, compared with the aminoacid sequence of Clover, only has following three mutational sites: N149Y, G160C and A206K.The green fluorescent protein that the present invention names this new is mClover3, particularly, its aminoacid sequence see shown in SEQIDNo.4 (wherein, the 150th of the corresponding SEQIDNo.4 aminoacid sequence in described N149Y mutational site, the 161st of the corresponding SEQIDNo.4 aminoacid sequence in described G160C mutational site, the 207th of the corresponding SEQIDNo.4 aminoacid sequence in described A206K mutational site), the gene order of this albumen of preferred coding mClover3 of the present invention's design is see shown in SEQIDNo.3.
New green fluorescent protein of the present invention, in vivo, the transformation period of dClover2 light stability in brightness normalization method situation is 98 seconds, mClover3 in vivo time in brightness normalization method situation the transformation period of light stability be 80 seconds, and Clover only has 50 seconds.
On the other hand, present invention also offers the fusion rotein of described green fluorescent protein, such as, the fusion rotein of mClover3 and mRuby3.By systematically testing mClover3 in mammal cell line, the fusion rotein demonstrating mClover3 can combine with the important subcellular object region in mammal cell line accurately, mClover3 with mNeonGreen compares and has higher transferring efficiency of fluorescence resonance energy with mEGFP, the efficiency of mClover3-mRuby3 and mNeonGreen-mRuby3 is also high than Clover-mRuby3, mClover3 and mNeonGreen is the most effective donor of mRuby3.
On the other hand, present invention also offers described red fluorescent protein as the application in FRET (fluorescence resonance energy transfer) acceptor.Particularly, wherein, the donor of described FRET (fluorescence resonance energy transfer) can be selected from following albumen: mEGFP, Envy, mNeonGreen, Clover or new green fluorescent protein of the present invention.
Present invention also offers described green fluorescent protein as the application in FRET (fluorescence resonance energy transfer) donor.Particularly, wherein, the acceptor of described FRET (fluorescence resonance energy transfer) is selected from following albumen: mCherry, mKate2, FusionRed, mRuby2 or mRuby3.
Present invention also offers a kind of albumen pair that can be used for FRET (fluorescence resonance energy transfer) imaging, it comprises:
New red fluorescent protein of the present invention is as FRET (fluorescence resonance energy transfer) acceptor; And/or
New green fluorescent protein of the present invention is as FRET (fluorescence resonance energy transfer) donor.
Present invention also offers a kind of genes encoding biosensor that can be used for optical imagery, this biosensor comprises:
New red fluorescent protein of the present invention is as FRET (fluorescence resonance energy transfer) acceptor; And/or
New green fluorescent protein of the present invention is as FRET (fluorescence resonance energy transfer) donor.
Compared with the biosensor of the genes encoding generally used, sensor of the present invention effectively can be lowered into picture background noise in fluorescence imaging process, significantly improves the sensitivity of fluorescence imaging, makes imaging more clear.Light stability due to this sensor has had and has greatly improved, and effectively extending can imaging time, makes to observe certain reaction for a long time or phenomenon is more prone to.Due to the phototoxic reduction of this sensor, alleviate on the normal reaction of cell and the impact of function in experimentation, experiment knot error is reduced, and experimental result is more pressed close to truly.
Particularly, in a specific embodiments of the present invention, construct respectively with Clover and mRuby3 as the right Camui α sensor Camui α-CR3 of FRET, mClover3 and mRuby3 is the Camui α sensor Camui α-C3R3 that FRET is right, mNeonGreen and mRuby3 is the Camui α sensor Camui α-NR3 that FRET is right, in Camui α telltale, new redness and green fluorescent protein are in the ability improving FRET baseline in test, and result display mClover3-mRuby3 and mNeonGreen3-mRuby3 improves reaction effect in Camui α.
On the other hand, the present invention further developed a set of assessment fluorescin criteria Assessment as independent marking and genes encoding biosensor in mammal cell line, the fluorescence signal intensity produced by the expression of this appraisal procedure inspection fluorescin derivative in mammal cell line.
The present invention has 5 large advantages compared with the biosensor of the genes encoding generally used: (1) chooses green and red fluorescent protein merges mutually, original cyan is replaced to merge mutually with yellow fluorescence protein, arrange in pairs or groups mutually with the fluorophore of green and red two kinds of colors, the distance of spectral separation is made to become large, alleviate interference, improve sensitivity.(2) there is great raising in the light stability of fluorescin, and fluorescin mClover3 improves 60% compared to last generation Clover.And mRuby3 improves 200% compared to last generation mRuby2, mRuby3 albumen is made to become the strongest monomer red fluorescent protein of light stability so far.(3) luminance brightness of fluorescin is also greatly improved, and such as mRuby3, than last generation mRuby2 bright 35%, makes mRuby3 become the red fluorescent protein that luminance brightness is the brightest so far.(4) high expression level, after transfection mammalian cell system, highly sensitive in the biosensor generally used now to cell imaging.(5) mRuby3 is the good probe detecting albumen in time-lapse shooting imaging and the restricted situation of photon, comprises quick time-lapse shooting imaging and single molecular imaging.
Accompanying drawing explanation
Fig. 1 a shows sequence and the comparing result of mRuby3 and mRuby2 prlmary structure of protein.
Fig. 1 b is the sudden change contrast crystalline structure figure of mRuby3 and mRuby2.
Fig. 1 c is that the absorption figure (left side) of red fluorescent protein mCherry, mKate2, FusionRed, mRuby2 and mRuby3 schemes on (right side) with transmitting.
Fig. 1 d show mRuby3, mRuby2 and mRuby2-M160I mutant cultivate in bacteriological incubator after fluorogram.
Fig. 2 a is the fluorescence imaging figure to specific subcellular structure after HeLa cells mRuby3 fusion rotein.From left to right: mRuby3-7aa-actin (actin cytoskeleton), mRuby3-6aa-tubulin (microtubule), connexin43 (cell adhesion contact)-7aa-mRuby3, mRuby3-10aa-H2B (nucleosome).Middle aa represents the unit of the distance between connection two albumen, is 1 aa with single amino acids distance.
Fig. 2 b is presented at four kinds of monomer red fluorescent protein mCherry in HEK293A and HeLa cell, and the luminance factor of FusionRed, mRuby2 and mRuby3 comparatively.
Fig. 3 a shows sequence and the comparing result of the prlmary structure of protein of mClover3, dClover2 and Clover.
Fig. 3 b is the crystalline structure figure that mClover3 and Clover compares mutational site.
Fig. 3 c is the absorption (left side) of green fluorescent protein mEGFP, Envy, mNeonGreen, Clover and Clover3 and launches (right side) spectrogram.
Fig. 4 a is the fluorescence imaging figure to specific subcellular structure after HeLa cells mClover3 fusion rotein.From left to right: mClover3-7aa-actin (actin cytoskeleton), mClover3-6aa-tubulin (microtubule), connexin43 (cell adhesion contact)-7aa-mClover3, mClover3-10aa-H2B (nucleosome).Middle aa represents the unit of the distance between connection two albumen, is 1 aa with single amino acids distance.
Fig. 4 b is presented at by expressing GFP-P2A-mCherry in HEK293A and HeLa cell, to the luminance factor of six kinds of monomer green fluorescent proteins comparatively.
Fig. 4 c is the efficiency comparative figure of the FRET (fluorescence resonance energy transfer) of three kinds of GFP-mRuby3 albumen in HEK293A and HeLa cell.
Fig. 5 a shows the right structure of the green/red FRET that is connected with Camui α.
Fig. 5 b shows the emission ratios (RDA) that green/red expresses HeLa cell donor/acceptor when not having Calcium ionophore to stimulate of Camui α.Data show with means standard deviation form.
In Fig. 5 c, left picture is that the emission ratios (RDA) of average donor/acceptor is schemed over time, and right picture is the HeLa cell volume efficiency figure that reaction obtains under Calcium ionophore stimulates.The change of the emission ratios of each cell represents by grey lines in the drawings, and mean value black line represents.Data show with means standard deviation form.Camui α-CR there is significant difference with Camui α-C3R3 and Camui α-NR3 exciting the ratio at peak value place respectively.
Embodiment
In order to more clearly understand the present invention, further describe the present invention referring now to the following example and accompanying drawing.Embodiment does not only limit the present invention in any way for explanation.In following experiment, the all commercially available acquisition of each Starting reagents material, the experimental technique of unreceipted actual conditions comprises ordinary method and normal condition that site-directed mutagenesis technique, fusion protein technology, artificial liposome cell transfection technique etc. are affiliated known, or according to the condition that apparatus manufacturer is advised.
During the present invention respectively tests, major technique used and experimental program comprise:
(1) distance model of FRET (fluorescence resonance energy transfer).
The formula of the model set up is
represent the fluorescence intensity at a certain wavelength place.ε
d(λ
ex) represent the optical extinction coefficient of donor under excitation wavelength.E represents transferring efficiency of fluorescence resonance energy.
it is the quantum yield representing donor.F
d(λ) the standardized utilizing emitted light at af at wavelength lambda donor is represented.ε
a(λ
ex) be the optical extinction coefficient representing acceptor under excitation wavelength.And Internal Fluorescent group distance (interfluorophoredistance, r) by Foster (
) formula E=1/ (1+ (r
6/ r
0 6) calculate, r
0for Foster (
) radius.
(2) construction of recombinant plasmid
Plasmid construction comprises: polymerase chain reaction (PCR), overlap-extension polymerase chain reaction (overlapPCR), and restriction endonuclease reaction is connected with In-Fusion, transforms DH5 α bacterium.All recombinant vectorss are by order-checking qualification.
(3) HeLa and HEK293A cell cultures and transfection
Utilize lipofection to be proceeded to by recombinant plasmid in HeLa and HEK293A cell, after 3-5 hour, change nutrient solution, to cultivate after cell dissociation after 24 hours, be dispensed in 8 orifice plates, then cultivate 24 hours.
(4) fluorescence microscope fusion rotein
For mClover3-mRuby3 fusion rotein, being transfected in HeLa cell after 24-72 hour, utilize the imaging of FV1000 laser confocal microscope.For mClover3, excite under 488nm exciting light, collect the light of 500-600nm.For mRuby3, excite at 559nm place, collect the light at 570-670nm place.The image software I mageJ process obtained.
(5) the external feature of fluorescin
N-end due to fluorescin is connected with six Histidines, so use cobalt resin purification (HisPurCobaltResin).And absorbancy, excitation spectrum and emmission spectrum are measured by multi-functional microplate reader SafireII.(for each protein sample, prepare just the same two parts of solution, protein dissolution is in PBS by denaturation method for optical extinction coefficient.A copy of it measures absorption peak, portion measures absorption peak under 1NNaOH effect in addition, optical extinction coefficient is calculated by formula ' before sex change/sex change after * 44000 ') measure, quantum yield then with Clover and mRuby2 for reference to (measuring emission spectrum and the absorption spectrum of testing sample and reference protein respectively, then the absorption value of emission spectrum area and acquisition emission spectrum is calculated respectively, then by emission spectrum area divided by absorption value, then by the ratio of the above-mentioned ratio of testing sample divided by reference protein, then the quantum yield of reference protein is multiplied by, the quantum yield of testing protein can be learnt).
In photobleaching experiment in vitro, with the protein of purifying for sample, use inverted fluorescence microscope oil mirror, use 40 × 0.90-NAUPlanS-Apo object lens, use X-Cite120-w metal halogen lamp at 100% neutral density respectively through excitation filter (for mRuby mutant) and a 485/30nm excitation filter (for Clover mutant) of a 545/30nm.Under metal chloride light source Continuous irradiation, within every 1 second, gather an image (camera is ORCA-ERCCD), multiple is also adjusted to and produces photon output rate is 1000 photons per sec.
Plural gel post (Superdex20030/100GLcolumn) is used in gel permeation chromatography experiment.Applied sample amount is 100 μ L, and concentration is 10 μMs.Wash-out mobility is 0.5 ml/min.By the absorption monitoring albumen wash-out at 280nm place.
(6) measurement of basic fluorescence Resonance energy transfer
GFP-RFP fusion rotein holds the avGFP redundant organism that blocks or mNeonGreen to merge the aa3-233 of mRuby2 or mRuby3 by catenation sequence by C-.Fusion rotein is transfected in HEK293A and HeLa cell.After transfection 2 days, cell is transferred in 96 orifice plates and detect fluorescence spectrum.Emmission spectrum scope is 490-750nm, excites with 470nm.
(7) in mammal cell line, compare the luminance brightness of mutant
When the mutant of mammal cell line Green fluorescin and mRuby is compared, with mCherry and mTurquoise2 for expressing internal reference, use liposome transfection recombinant plasmid in HEK293A and HeLa cell, after transfection 2 days, cell is transferred in 96 transparent orifice plates of bottom and detect fluorescence spectrum.The parameters of different fluorescins is as follows: mTurquoise2-434/5nm-474/5nm, mCherry-587/20nm-610/5nm, GFP-430/20nm-480 ~ 650nm, RFP-550/10nm-570 ~ 670nm.Relative brightness is that the green fluorescent protein integrated or mRuby mutant are launched light intensity and obtained divided by the transmitting light intensity of mCherry or mTurquoise2.
(8) Camui α sensor improves and feature
In order to build Camui α-CR mutant, N-end is that the junctor (between GFP and CaMKII α) of NheI restriction enzyme site and a C-end extended coding is used to increase the avGFP (not containing ' GITHGMDELYK ' sequence) of C-end brachymemma or mNeonGreen (not containing ' GMDELYK ' sequence).After the CaMKII α region of any one end is successful by pcr amplification, to mRuby2 or mRuby3 also by PCR method amplification object fragment, the junctor of a N-end expansion is introduced in reaction simultaneously in CaMKII α region, introduce ApaI restriction enzyme site at C-end, by overlap-extension polymerase chain reaction, Insert Fragment is cloned on carrier pcDNA3.1.
After the reorganized plasmid transfection of cell 2 days, use microscope observing cell.Use cold ORCA-ERCCD pick up camera and 40 × 1.2-NAC-Apochromatic mirror water logging modular view of inverted fluorescence microscope 200M, use software Micro-manager1.4, design parameter is: 17-inch2.5-GHzCore2DuoMacBookProrunningMacOS10.6.8.Continuously FRET and donor emission imaging is obtained by following spectral filter: green fluorescent protein excites HQ470/30nm for exciting HQ470/30nm and launching 505AELPnm, FRET use and launches BA575IFnm.
(9) Ratio-type image analysis
The measurement of FRET is quantized by software I mageJ.Source document is 16-position tiff file, selects the visual field with random method, and transfection sends fluorocyte for positive, with the cell of untransfected for background detection.The emissive porwer that background removes donor is emission ratios divided by the value that the intensity that background removes FRET obtains.Use a full spectrum look-up table (minimum value is blue, and maximum value is red), produce intensity adjustments display by receptor channel.
(10) statistical method
Variance analysis and Deng Nite method posteriority is adopted to detect the difference determining brightness measured in cell.The peak emission ratio of Camui alpha-mutant changes whether have significant difference to adopt T inspection to determine.Mapping software is Excel and Prism.
Embodiment 1: the fluorescin pair that can be used for highly sensitive FRET imaging
The character of 1 new red fluorescent protein mRuby3 and imaging effect
The structure of 1.1mRuby3 and photophysical property
Adopt site-directed mutagenesis technique on mRuby2 basis, carry out site-directed mutagenesis and obtain new red fluorescent protein mRuby3 of the present invention.The aminoacid sequence of mRuby3 is see shown in SEQIDNo.2, see shown in SEQIDNo.1, (method for synthesizing gene by PCR-based polymerization obtains the DNA sequence dna of mRuby3 to the gene order of this albumen of preferred coding mRuby3 of the present invention's design, namely a lot of PCR primer is designed, there is the overlap of 18 ~ 25bp in every two primers, then by the method for overlapPCR by all primer-oligomerizations, thus the complete gene of stroke one).
The mRuby2 of mRuby3 and previous generation compares aminoacid sequence 21 replacements, is specially: N33R, M36E, T38V, K74A, G75D, M105T, C114E, H118N, Q120K, H159D, M160I, S171H, S173N, I192V, L202I, M209T, F210Y, H216V, F221Y, A222S, G223N, can see Fig. 1 a, and the crystalline structure of mRuby (PDB database ID is 3U0M) can see Fig. 1 b.In Fig. 1 a, the amino acid forming chromophore marks by black box; The sudden change blueness being positioned at albumen outer wall marks, and comprises N33R, T38V, M105T, C114E, H118N, Q120K, H159D, S171H, S173N, L202I, F210Y, H216V; The sudden change green being positioned at inwall marks, and comprising: M160I; The sudden change being positioned at ring place marks with orange, comprising: M36E, K74A, G75D, I192V, M209T, F221Y, A222S, G223N.
The absorption figure of red fluorescent protein mCherry, mKate2, FusionRed, mRuby2 and mRuby3 can see figure left in Fig. 1 c, and each protein emission figure can see figure right in Fig. 1 c.Visible in figure, respectively at 558nm and 592nm place, there is blue shift in the excitation spectrum of mRuby3 and the peak value of emmission spectrum compared with mRuby2.Optical extinction coefficient at peak value place is 128mM
-1cm
-1, quantum yield is 0.45 (see table 1), so the light strength ratio mRuby2 of mRuby3 exceeds 35%, so it is monomer red fluorescent protein the brightest so far.In addition, the light stability of mRuby3 is fine, and under the irradiation of Jupiter, the transformation period of mRuby3 is 349 seconds, is longer than 123 seconds of mRuby2 and 337 seconds of TagRFP-T.In photobleaching kinetics, mRuby3 shows single index relation, and its dissociation constant value is 4.8, and acid resistance is similar compared with mRuby2.So mRuby3 to be brightness so far the brightest and light stablizes best red fluorescent protein.
Table 1: the photophysical property of monomer green and red fluorescent protein
The wavelength at place when table note: b is exciting light peak value.The wavelength at place when c is utilizing emitted light peak value.D is maximum optical extinction coefficient.E is fluorescence quantum yield.F is the relative value of opalescence brightness.G is dissociation constant.H is the light stability of albumen, and representing that under Jupiter irradiates each molecule is per second is 500 photons time used from 1000 photon photobleachings, and time unit is second.
In each mutational site that mRuby3 and mRuby2 compares, M160I makes albumen brighter, but protein maturation or folding variation; Other site then allows protein maturation or folding to improve.In the present invention, also on the basis of mRuby2, rite-directed mutagenesis obtains the mutant only compared with mRuby2 with M160I mutational site, called after mRuby2-M160I in the present invention.Picture (1) in Fig. 1 d shows the fluorogram of mRuby2 (the right) and mRuby2-M160I (left side) mutant incubated overnight 24h in bacteriological incubator, and the picture (2) in Fig. 1 d shows the fluorogram that mRuby2 (the right) and mRuby2-M160I (left side) mutant cultivate 48h in bacteriological incubator.As we know from the figure, mRuby2-M160I is due to ripe or folding slow, and therefore 24h is darker than mRuby2 after cultivating, but along with the prolongation of incubation time, finally bright than mRuby2.Picture (3) in Fig. 1 d is the fluorogram of mRuby3 and mRuby2 in bacteriological incubator after incubated overnight 24h (in figure, the left side is mRuby3, and the right is mRuby2).
1.2mRuby3 is imaging in mammal cell line
The luminance brightness of mutant is compared in mammal cell line.Fig. 2 a is the fluorescence imaging figure to specific subcellular structure after HeLa cells mRuby3 fusion rotein.From left to right: mRuby3-7aa-actin (actin cytoskeleton), mRuby3-6aa-tubulin (microtubule), connexin43 (cell adhesion contact)-7aa-mRuby3, mRuby3-10aa-H2B (nucleosome).Middle aa represents the unit of the distance between connection two albumen, is 1 aa with single amino acids distance.Demonstrate fusion rotein (the pcr amplification acquisition mRuby3 sequence of mRuby3, then this is connected in the pEGFP-C1 carrier of EcoRI and BglII double digestion and build pmRuby3-C1, then subcellular addressing sequence is appealed in amplification respectively, is then connected into construction of fusion protein in the pmRuby3-C1 carrier of BamHI and EcoRI double digestion by in-fusion technology) can combine with the important subcellular object region in mammal cell line accurately.
The red fluorescent protein of the fluorescent signal then produced in mammalian cell by mRuby3 and other monomer compares.Fig. 2 b shows the luminance factor of four kinds of monomer red fluorescent proteins mCherry, FusionRed, mRuby2 and mRuby3 in HEK293A and HeLa cell comparatively, the strength of signal of result display mRuby3 expressed by HEK293A and HeLa cell is the strongest, exceeds at least 100% (Fig. 2 b) than red fluorescent protein mRuby2, FusionRed and mCherry.The result that this detection method of the present invention obtains is higher with independent each molecule luminance brightness detection method phase specific sensitivity.
As can be seen here, mRuby3 is the outstanding acceptor of a FRET in theory.Because mRuby2 has proved an efficient FRET (fluorescence resonance energy transfer) acceptor.So compared by mRuby3 and mRuby2, with identical green fluorescent protein Clover for donor, find that mRuby3 is more efficient than mRuby2.
The character of 2 new green fluorescin mClover3 and imaging effect
The structure of 2.1mClover3 and photophysical property
Equally also transform green fluorescent protein Clover in the present invention, intention obtains a luminance brightness and light stability and to promote to some extent and can as the albumen of the efficient donor of mRuby3 FRET (fluorescence resonance energy transfer).(the GeneMorphIIRandomMutagenesis test kit of Clontech company is adopted to carry out by random mutation technology, ensure 1 ~ 3bp sudden change/1000bp) obtain the mutant of Clover, then by mutant that the irradiation screening light stability of blue LED is strong.Clover1.5 (the 149th becomes tyrosine by l-asparagine) is obtained, dClover2 (the 149th becomes tyrosine by aspartic acid, and the 160th becomes Serine by glycine) through screening.In vivo, the transformation period of dClover2 light stability in brightness normalization method situation is 98 seconds, and Clover only has 50 seconds.Optical extinction coefficient is 123mM
– 1cm
– 1,also than the 111mM of Clover
– 1cm
– 1increase.Relative quantum yields is 0.8 slightly higher than 0.76 of Clover.Further across the 206th site L-Ala being become Methionin and after 160 site Serines are become halfcystine, obtaining the fluorescin mClover3 of monomer on the basis of dClover2.The aminoacid sequence of mClover3 is see shown in SEQIDNo.4, and the gene order of this albumen of preferred coding mClover3 of the present invention's design is see (method by gene chemical synthesis obtains the DNA sequence dna of mClover3) shown in SEQIDNo.3.
Fig. 3 a shows sequence and the comparing result of the prlmary structure of protein of mClover3, dClover2 and Clover.The amino acid forming chromophore marks by black box.Three places' sudden change (N149, G160, A206) be positioned on albumen wall mark with orange.Fig. 3 b shows the crystalline structure that mClover3 and Clover compares mutational site.Fig. 3 c shows absorption spectrum (left figure) and the emmission spectrum (right figure) of green fluorescent protein mEGFP, Envy, mNeonGreen, Clover and Clover3, can find out that mClover3 and Clover compares and have similar spectrum, and also similar in brightness.Another mClover3 in vivo time in brightness normalization method situation the transformation period of light stability be 80 seconds.
2.2mClover3 is imaging in mammal cell line
In mammal cell line, mClover3 is systematically tested.
Fig. 4 a shows the fluorescence imaging figure to specific subcellular structure after HeLa cells mClover3 fusion rotein.From left to right: mClover3-7aa-actin (actin cytoskeleton), mClover3-6aa-tubulin (microtubule), connexin43 (cell adhesion contact)-7aa-mClover3, mClover3-10aa-H2B (nucleosome).Middle aa represents the unit of the distance between connection two albumen, is 1 aa with single amino acids distance.The fusion rotein demonstrating mRuby3 can combine with the important subcellular object region in mammal cell line accurately.The green fluorescent protein of the fluorescent signal then produced in mammalian cell by mRuby and mRuby3 and other monomer compares, as mEGFP, sfGFP and Envy.MEGFP is present purposes fluorescin the most widely.SfGFP is the redundant organism with high folding efficiency of mEGFP.And Envy in budding yeast, be considered to brightness maximum.But these three kinds of albumen all do not have Clover and mClover3 brightness large (table 1) in vitro in extract.But in mammal cell line, show similar brightness.And the monomer mNeonGreen reported recently compare in vitro mClover3 want bright 10% (table 1), the two has similar excitation and emission spectra, having occurred a little red shift compared with green fluorescent protein (GFP), may be that cation-π interaction has appearred in fluorescence chromophore.
By expressing GFP-P2A-mCherry in HEK293A and HeLa cell, compare the brightness of six kinds of monomer green fluorescent proteins, result is see Fig. 4 b.Find in HEK293A cell, the cell average fluorescent strength of transfection mNeonGreen is the brightest, and being secondly Clover or mClover3, is Envy again, that the most weak is sfGFP.Difference between mNeonGreen and mClover3, mClover3 and EGFP or sfGFP has statistical significance (p<0.05).In HeLa cell, there is very strong fluorescence after expressing in mNeonGreen, Clover or mClover3, and difference in brightness is little each other, and the brightness of Envy is taken second place, and is then EGFP, is finally sfGFP.Luminance difference between mClover3, Envy, EGFP and sfGFP has statistical significance.
In order to find the optimal monomer green fluorescent protein that can do the best donor of FRET (fluorescence resonance energy transfer) of mRuby3, the green fluorescent protein mClover of the monomer that will detect, mClover3, mNeonGreen and EGFP and mRuby3 makes fusion rotein, then in mammalian cell, detect the efficiency of the FRET (fluorescence resonance energy transfer) of various fusion rotein, the results are shown in Figure 4c, the emissioning light spectrum (black line) of emissioning light spectrum (red line) and the linear fit of display experiment gained coincideing very, mClover3 with mNeonGreen compares and has higher transferring efficiency of fluorescence resonance energy with mEGFP.The efficiency of mClover3-mRuby3 and mNeonGreen-mRuby3 also higher than Clover-mRuby3 (Fig. 4 c).As can be seen here, mClover3 and mNeonGreen is the most effective donor of mRuby3.
3Camui α sensor
First build respectively with Clover and mRuby3 for the right Camui α-CR3 of FRET, mClover3 and mRuby3 is the Camui α-C3R3 that FRET is right, mNeonGreen and mRuby3 is the Camui α-NR3 that FRET is right.
Fig. 5 a shows the right structure of the green/red FRET that is connected with Camui α.Experiment proves that Clover3-mRuby3 improves the sensitivity of FRET (fluorescence resonance energy transfer) when detecting CaMKII α active.In Camui α tests, fluorescin merges mutually with the terminal of CaMKII α polypeptide and forms FRET couple.Consistent with structural analysis, Camui α FRET ability under disabled state is high, and low (Fig. 5 a) for FRET ability in an active state.
Then new redness and green fluorescent protein is tested in Camui α telltale in the ability improving FRET baseline.Fig. 5 b shows the emission ratios (RDA) that green/red expresses HeLa cell donor/acceptor when not having Calcium ionophore to stimulate of Camui α, and the FRET baseline of the Camui α-CR3 of display containing mRuby3 is than the Camui α-CR high (Fig. 5 b) containing mRuby2.Only Clover is replaced with mClover3 or mNeonGreen and significantly can not change basic FRET level (Fig. 5 b).Data show with means standard deviation form.
The appearance that finally whether the effect of detection Camui α own can be right because of new FRET promotes to some extent.Under the stimulation of Calcium ionophore, in HeLa cell, compare the reaction of Camui α-CR ,-CR3 ,-C3R3 and-NR3.The left figure of Fig. 5 c is that the emission ratios (RDA) of average donor/acceptor is schemed over time, and right figure is that HeLa cell reacts the volume efficiency figure obtained under Calcium ionophore stimulates.The change of the emission ratios of each cell represents by grey lines in the drawings, and mean value black line represents.Data show with means standard deviation form.Camui α-CR there is significant difference with Camui α-C3R3 and Camui α-NR3 exciting the ratio at peak value place respectively.The average response of Camui α-CR3 is similar to Camui α-CR, greatly about 45% (Fig. 5 c).If mClover3 and mNeonGreen is replaced Clover will improve Camui α dynamicrange significantly, the enhancing of calcium induction improves green/red emission ratios.In Camui α-CR3, improve 45%, in Camui α-C3R3, improve 70%, in Camui α-NR3, improve 56% (Fig. 5 c).Although Camui α-CR3 ,-C3R3 have similar r with-NR3
0value and FRET baseline, but there is significant lifting in the susceptibility of Camui α.If mNeonGreen or mClover3 has occurred that 149 become Y and the 206th by N and become K two positions by A and suddenly change, and can promote that Camui α-C3R3 and Camui α-NR3 changes to active state.
Claims (10)
1. a red fluorescent protein, the aminoacid sequence of this red fluorescent protein, compared with the aminoacid sequence of mRuby2, has following mutational site: M160I;
Preferably, the aminoacid sequence of this red fluorescent protein, compared with the aminoacid sequence of mRuby2, also has following mutational site: N33R, M36E, T38V, K74A, G75D, M105T, C114E, H118N, Q120K, H159D, S171H, S173N, I192V, L202I, M209T, F210Y, H216V, one or more combination in F221Y, A222S, G223N.
2. red fluorescent protein according to claim 1, it is for being selected from the albumen of following (a) or (b):
A () has the albumen of aminoacid sequence as shown in SEQIDNo.2;
Through replacing, lacking or add one or several amino acid and with (a), there is the albumen derivative by (a) of identical function in b aminoacid sequence that () limits at (a).
3. a green fluorescent protein, the aminoacid sequence of this green fluorescent protein, compared with the aminoacid sequence of Clover, has following mutational site: N149Y;
Preferably, the aminoacid sequence of this green fluorescent protein, compared with the aminoacid sequence of Clover, also has following mutational site: G160S or G160C;
More preferably, the aminoacid sequence of this green fluorescent protein, compared with the aminoacid sequence of Clover, also has following mutational site: A206K.
4. green fluorescent protein according to claim 3, it is for being selected from the albumen of following (a) or (b):
A () has the albumen of aminoacid sequence as shown in SEQIDNo.4, SEQIDNo.5 or SEQIDNo.6;
Through replacing, lacking or add one or several amino acid and with (a), there is the albumen derivative by (a) of identical function in b aminoacid sequence that () limits at (a).
5. the polynucleotide sequence of the red fluorescent protein of coding described in claim 1 or 2 or the green fluorescent protein described in claim 3 or 4;
Preferably, described polynucleotide sequence has sequence as shown in SEQIDNo.1 or SEQIDNo.3.
6. a fusion rotein, it comprises the red fluorescent protein described in claim 1 or 2 or the green fluorescent protein described in claim 3 or 4.
7. the red fluorescent protein described in claim 1 or 2 is as the application in FRET (fluorescence resonance energy transfer) acceptor;
Preferably, wherein, the donor of described FRET (fluorescence resonance energy transfer) is selected from following albumen: mEGFP, Envy, mNeonGreen, Clover or the albumen described in claim 3 or 4.
8. the green fluorescent protein described in claim 3 or 4 is as the application in FRET (fluorescence resonance energy transfer) donor;
Preferably, wherein, the acceptor of described FRET (fluorescence resonance energy transfer) is selected from following albumen: mCherry, mKate2, FusionRed, mRuby2 or mRuby3.
9. one kind can be used for the albumen pair of FRET (fluorescence resonance energy transfer) imaging, and it comprises:
Red fluorescence egg described in claims 1 or 2 is as FRET (fluorescence resonance energy transfer) acceptor; And/or
Green fluorescent protein described in claim 3 or 4 is as FRET (fluorescence resonance energy transfer) donor.
10. can be used for a genes encoding biosensor for optical imagery, this biosensor comprises:
Red fluorescent protein described in claims 1 or 2 is as FRET (fluorescence resonance energy transfer) acceptor; And/or
Green fluorescent protein described in claim 3 or 4 is as FRET (fluorescence resonance energy transfer) donor.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910057635.8A CN109762050B (en) | 2015-12-31 | 2015-12-31 | Fluorescent protein pair for high-sensitivity FRET imaging and application thereof |
| CN201511025610.8A CN105504027B (en) | 2015-12-31 | 2015-12-31 | Fluorescent protein pair for high-sensitivity FRET imaging and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201511025610.8A CN105504027B (en) | 2015-12-31 | 2015-12-31 | Fluorescent protein pair for high-sensitivity FRET imaging and application thereof |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910057635.8A Division CN109762050B (en) | 2015-12-31 | 2015-12-31 | Fluorescent protein pair for high-sensitivity FRET imaging and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105504027A true CN105504027A (en) | 2016-04-20 |
| CN105504027B CN105504027B (en) | 2020-08-07 |
Family
ID=55712380
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201511025610.8A Active CN105504027B (en) | 2015-12-31 | 2015-12-31 | Fluorescent protein pair for high-sensitivity FRET imaging and application thereof |
| CN201910057635.8A Active CN109762050B (en) | 2015-12-31 | 2015-12-31 | Fluorescent protein pair for high-sensitivity FRET imaging and application thereof |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910057635.8A Active CN109762050B (en) | 2015-12-31 | 2015-12-31 | Fluorescent protein pair for high-sensitivity FRET imaging and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (2) | CN105504027B (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106632640A (en) * | 2017-02-24 | 2017-05-10 | 中国科学院深圳先进技术研究院 | Red fluorescence protein, fusion protein, separated nucleic acid, carrier and application |
| CN106831971A (en) * | 2017-02-24 | 2017-06-13 | 中国科学院深圳先进技术研究院 | A kind of remote red fluorescent protein, fusion protein, separate nucleic acid, carrier and application |
| CN108503701A (en) * | 2017-02-24 | 2018-09-07 | 中国科学院深圳先进技术研究院 | A kind of fluorescin, fusion protein, the nucleic acid of separation, carrier and application |
| CN108659110A (en) * | 2017-04-01 | 2018-10-16 | 北京义翘神州科技有限公司 | A kind of improved red fluorescent protein and its application |
| CN113201058A (en) * | 2021-05-06 | 2021-08-03 | 深圳先进技术研究院 | Green fluorescent protein Clover4, bioluminescence resonance energy transfer-based probe derived from green fluorescent protein Clover4 and application of bioluminescence resonance energy transfer-based probe |
| CN114813706A (en) * | 2022-06-29 | 2022-07-29 | 国科大杭州高等研究院 | Blood cell hyperspectral optical tweezers capture energy resonance transfer analyzer |
| WO2022232983A1 (en) * | 2021-05-06 | 2022-11-10 | 深圳先进技术研究院 | Green fluorescent protein clover4, probe derived from green fluorescent protein clover4 and based on bioluminescence resonance energy transfer, and application of green fluorescent protein clover4 |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112961225B (en) * | 2019-12-14 | 2022-08-19 | 深圳先进技术研究院 | Near-infrared fluorescent protein, recombinant vector, recombinant cell and application thereof |
| CN115028743B (en) * | 2022-06-28 | 2024-05-14 | 山东大学 | Fluorescent sensor for detecting D-2-hydroxyglutarate and construction method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013151511A1 (en) * | 2012-04-05 | 2013-10-10 | Agency For Science, Technology And Research | Modified Dual-Colour Protein |
| WO2015039211A1 (en) * | 2013-09-17 | 2015-03-26 | Mcmaster University | Method for assaying a protease |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012063897A1 (en) * | 2010-11-12 | 2012-05-18 | 独立行政法人科学技術振興機構 | Modified fluorescent protein |
-
2015
- 2015-12-31 CN CN201511025610.8A patent/CN105504027B/en active Active
- 2015-12-31 CN CN201910057635.8A patent/CN109762050B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013151511A1 (en) * | 2012-04-05 | 2013-10-10 | Agency For Science, Technology And Research | Modified Dual-Colour Protein |
| WO2015039211A1 (en) * | 2013-09-17 | 2015-03-26 | Mcmaster University | Method for assaying a protease |
Non-Patent Citations (1)
| Title |
|---|
| AMY LAM ET AL: "Improving FRET dynamic range with bright green and red fluorescent proteins", 《NAT METHODS》 * |
Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106831971B (en) * | 2017-02-24 | 2020-10-27 | 中国科学院深圳先进技术研究院 | Far-red fluorescent protein, fusion protein, isolated nucleic acid, vector and application |
| CN106831971A (en) * | 2017-02-24 | 2017-06-13 | 中国科学院深圳先进技术研究院 | A kind of remote red fluorescent protein, fusion protein, separate nucleic acid, carrier and application |
| CN108503701A (en) * | 2017-02-24 | 2018-09-07 | 中国科学院深圳先进技术研究院 | A kind of fluorescin, fusion protein, the nucleic acid of separation, carrier and application |
| CN106632640A (en) * | 2017-02-24 | 2017-05-10 | 中国科学院深圳先进技术研究院 | Red fluorescence protein, fusion protein, separated nucleic acid, carrier and application |
| CN106632640B (en) * | 2017-02-24 | 2020-04-14 | 中国科学院深圳先进技术研究院 | Red fluorescent protein, fusion protein, isolated nucleic acid, vector and application |
| CN108503701B (en) * | 2017-02-24 | 2020-07-24 | 中国科学院深圳先进技术研究院 | Fluorescent protein, fusion protein, isolated nucleic acid, vector and application |
| CN108659110A (en) * | 2017-04-01 | 2018-10-16 | 北京义翘神州科技有限公司 | A kind of improved red fluorescent protein and its application |
| CN108659110B (en) * | 2017-04-01 | 2020-11-06 | 北京义翘神州科技股份有限公司 | Improved red fluorescent protein and application thereof |
| CN113201058A (en) * | 2021-05-06 | 2021-08-03 | 深圳先进技术研究院 | Green fluorescent protein Clover4, bioluminescence resonance energy transfer-based probe derived from green fluorescent protein Clover4 and application of bioluminescence resonance energy transfer-based probe |
| CN113201058B (en) * | 2021-05-06 | 2022-08-05 | 深圳先进技术研究院 | Green fluorescent protein Clover4, bioluminescence resonance energy transfer-based probe derived from green fluorescent protein Clover4 and application of bioluminescence resonance energy transfer-based probe |
| WO2022232983A1 (en) * | 2021-05-06 | 2022-11-10 | 深圳先进技术研究院 | Green fluorescent protein clover4, probe derived from green fluorescent protein clover4 and based on bioluminescence resonance energy transfer, and application of green fluorescent protein clover4 |
| CN114813706A (en) * | 2022-06-29 | 2022-07-29 | 国科大杭州高等研究院 | Blood cell hyperspectral optical tweezers capture energy resonance transfer analyzer |
| CN114813706B (en) * | 2022-06-29 | 2022-12-13 | 国科大杭州高等研究院 | Blood cell hyperspectral optical tweezers capture energy resonance transfer analyzer |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109762050B (en) | 2020-08-28 |
| CN105504027B (en) | 2020-08-07 |
| CN109762050A (en) | 2019-05-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105504027A (en) | Fluorescent protein for high-sensitivity FRET imaging and application thereof | |
| Rizzo et al. | Optimization of pairings and detection conditions for measurement of FRET between cyan and yellow fluorescent proteins | |
| Yang et al. | mBeRFP, an improved large stokes shift red fluorescent protein | |
| Nagai et al. | A high-throughput method for development of FRET-based indicators for proteolysis | |
| Kogure et al. | Fluorescence imaging using a fluorescent protein with a large Stokes shift | |
| Xu et al. | [12] Bioluminescence resonance energy transfer: Monitoring protein-protein interactions in living cells | |
| JP5570803B2 (en) | Fluorescent protein and pH measurement method | |
| WO2006037622A1 (en) | Screening process for the detection and characterization of protein-protein-interactions in vivo by fluorescence cross correlation spectroscopy | |
| US20090203035A1 (en) | Fluorescent proteins with increased photostability | |
| US20090191581A1 (en) | Cell-permeable fluorescent proteins | |
| Johnson et al. | Fluorescent protein applications in microscopy | |
| Meiresonne et al. | Detection of protein interactions in the cytoplasm and periplasm of Escherichia coli by Förster resonance energy transfer | |
| JP2011212008A (en) | FLUORESCENT PROTEIN AND METHOD FOR MEASURING pH | |
| Deo et al. | Bioluminescence resonance energy transfer from aequorin to a fluorophore: an artificial jellyfish for applications in multianalyte detection | |
| Periasamy et al. | FRET imaging of Pit-1 protein interactions in living cells | |
| JP5076037B2 (en) | Modified fluorescent protein | |
| US20140243509A1 (en) | Near infrared fluorogen and fluorescent activating proteins for in vivo imaging and live-cell biosensing | |
| Medine et al. | Time‐correlated single photon counting FLIM: Some considerations for physiologists | |
| CN106632640A (en) | Red fluorescence protein, fusion protein, separated nucleic acid, carrier and application | |
| JP5875773B2 (en) | Ultraviolet light measurement method | |
| KR102307241B1 (en) | Biosensor for detecting fucosyllactose and detecting method of fucosyllactose using the same | |
| Jyothikumar et al. | Förster Resonance Energy Transfer Microscopy for Monitoring Molecular Dynamics in Living Cells | |
| Salto González et al. | New Red-Emitting Chloride-Sensitive Fluorescent Protein with Biological Uses | |
| JP5796949B2 (en) | Fluorescent protein | |
| Lubbeck et al. | Microfluidic cytometer for simultaneous high-throughput screening of fluorescent proteins on the basis of fluorescence lifetime, photostability, and brightness |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB03 | Change of inventor or designer information |
Inventor after: Chu Jun Inventor after: Ring Michael Inventor after: Guo Yuqi Inventor after: Zhang Chuqiu Inventor after: Wang Huina Inventor before: Chu Jun Inventor before: Guo Yuqi Inventor before: Zhang Chuqiu Inventor before: Wang Huina |
|
| CB03 | Change of inventor or designer information | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |