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
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Abstract
本发明提供了可用于高灵敏FRET成像的荧光蛋白对及其应用。具体而言，本发明提供了一种红色荧光蛋白，该红色荧光蛋白的氨基酸序列与mRuby2的氨基酸序列相比，具有以下突变位点：N33R,M36E,T38V,K74A,G75D,M105T,C114E,H118N,Q120K,H159D,M160I,S171H,S173N,I192V,L202I,M209T,F210Y,H216V,F221Y,A222S,G223N。本发明还提供了一种绿色荧光蛋白，该绿色荧光蛋白的氨基酸序列与Clover的氨基酸序列相比，具有以下突变位点：N149Y,G160S或G160C,A206K。本发明的红色荧光蛋白和绿色荧光蛋白具有良好的光稳定性，可用于高灵敏FRET成像。The present invention provides fluorescent protein pairs that can be used for high-sensitivity FRET imaging and applications thereof. Specifically, the present invention 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, G223N. The present invention also 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, A206K. The red fluorescent protein and green fluorescent protein of the present invention have good photostability and can be used for high-sensitivity FRET imaging.
Description
技术领域technical field
本发明是关于可用于高灵敏FRET成像的荧光蛋白对及其应用。The present invention relates to pairs of fluorescent proteins that can be used for highly sensitive FRET imaging and their applications.
背景技术Background technique
荧光共振能量转移(fluorescenceresonanceenergytransfer,FRET)是距离很近的两个荧光分子间产生的一种能量转移现象。当供体荧光分子的发射光谱与受体荧光分子的吸收光谱重叠，并且两个分子的距离在10nm范围以内时，就会发生一种非放射性的能量转移，即FRET现象，使得供体的荧光强度比它单独存在时要低的多(荧光猝灭)，而受体发射的荧光却大大增强(敏化荧光)。而在生物体内，如果两个蛋白质分子的距离在10nm之内，一般认为这两个蛋白质分子存在直接相互作用。荧光共振能量转移技术能够实时监控活细胞内生物分子的活性和分子之间的相互反应，为研究复杂细胞信号通路提供了高时间和空间分辨率的有效方法。随着绿色荧光蛋白应用技术的发展，FRET已经成为检测活体中生物大分子纳米级距离和纳米级距离变化的有力工具，在生物大分子相互作用分析、细胞生理研究、免疫分析等方面有着广泛的应用。Fluorescence resonance energy transfer (fluorescence resonance energy transfer, FRET) is an energy transfer phenomenon between two fluorescent molecules that are very close to each other. When the emission spectrum of the donor fluorescent molecule overlaps with the absorption spectrum of the acceptor fluorescent molecule, and the distance between the two molecules is within 10nm, a non-radioactive energy transfer occurs, that is, the FRET phenomenon, so that the fluorescence of the donor The intensity is much lower than when it exists alone (fluorescence quenching), while the fluorescence emitted by the acceptor is greatly enhanced (sensitized fluorescence). In living organisms, if the distance between two protein molecules is within 10nm, it is generally considered that there is a direct interaction between the two protein molecules. Fluorescence resonance energy transfer technology can monitor the activity of biomolecules and the interaction between molecules in living cells in real time, and provides an effective method with high temporal and spatial resolution for the study of complex cell signaling pathways. With the development of green fluorescent protein application technology, FRET has become a powerful tool for detecting the nanoscale distance and nanoscale distance changes of biological macromolecules in vivo, and has a wide range of applications in biomacromolecule interaction analysis, cell physiology research, and immune analysis. application.
在生物光学与分子影像技术领域，科学家们一直在努力寻找高效的可用于光学成像的基因编码生物传感器。这些基因编码的生物传感器利用荧光共振能量转移技术实时的监控生物分子的活性和分子之间的相互反应。现在普遍使用的基因编码生物传感器是将青色和黄色荧光蛋白相融合，以青和黄两种颜色的荧光团相搭配，通过荧光共振能量转移实时监控生物分子的活性和分子之间的相互作用。但是这种生物传感器有诸多缺陷，主要表现在以下方面：(1)普通生物传感器中使用的青色和黄色两种荧光蛋白的动态感光范围较低，所以导致成像灵敏度低，很难对细胞内的一些瞬时和微弱生化反应进行监测。(2)光毒性大，在检测活的细胞或样品时，对细胞正常的代谢反应和分子相互作用产生较大的负面影响，导致实验结果出现较大的误差，细胞在长时间成像过程中可能死亡。(3)生物传感器自发荧光干扰。在细胞中成像时，由于传感器本身不可避免的激发青色荧光蛋白的同时可激发细胞内一些内源性分子，比如flavin，会出现自发荧光，所以对实验结果也会造成不同程度的干扰和影响。(4)在激发黄色荧光蛋白时青色荧光蛋白出现光活化。(5)传感器所使用的荧光蛋白对pH敏感，pH发生少许变化时，荧光蛋白可能会失活，荧光会明显减弱。In the field of bio-optics and molecular imaging technology, scientists have been working hard to find efficient gene-encoded biosensors that can be used for optical imaging. These gene-encoded biosensors use fluorescence resonance energy transfer technology to monitor the activity of biomolecules and the interaction between molecules in real time. The commonly used gene-encoded biosensors are the fusion of cyan and yellow fluorescent proteins, and the fluorophores of cyan and yellow are matched to monitor the activity of biomolecules and the interaction between molecules in real time through fluorescence resonance energy transfer. However, this biosensor has many defects, which are mainly manifested in the following aspects: (1) The cyan and yellow fluorescent proteins used in ordinary biosensors have a low dynamic sensitivity range, so the imaging sensitivity is low, and it is difficult to detect intracellular Some transient and weak biochemical reactions are monitored. (2) The phototoxicity is high. When detecting living cells or samples, it will have a large negative impact on the normal metabolic reactions and molecular interactions of cells, resulting in large errors in experimental results. Cells may be in the long-term imaging process. die. (3) Biosensor autofluorescence interference. When imaging in cells, since the sensor itself inevitably excites cyan fluorescent protein and at the same time excites some endogenous molecules in the cell, such as flavin, which will appear autofluorescence, so it will also cause different degrees of interference and influence on the experimental results. (4) The cyan fluorescent protein is photoactivated when the yellow fluorescent protein is excited. (5) The fluorescent protein used in the sensor is sensitive to pH. When the pH changes slightly, the fluorescent protein may be inactivated and the fluorescence will be significantly weakened.
2012年Lam,A.J.等人开发出来一个GFP-RFP对，两种荧光蛋白分别为Clover和mRuby2。该荧光蛋白FRET对与普通的CFP-YFPFRET对相比提高了反应的敏感性，降低了成像时光毒性，与其它的GFP-RFP对相比性质优越，且该方案也已经成功用于活细胞中Zn2+聚集和CaMKIIα活性的成像。但是不足之处为Clover的光稳定弱，在光连续照射下容易光漂白。而mRuby2的光强度也并不高，所以限制了该荧光蛋白FRET对的使用(Lam,A.J.etal.ImprovingFRETdynamicrangewithbrightgreenandredfluorescentproteins.NatMethods9,1005-1012(2012))。在此将该文献的全部内容并入于本发明中作为参考文献。In 2012, Lam, AJ et al. developed a GFP-RFP pair, and the two fluorescent proteins were Clover and mRuby2. Compared with the common CFP-YFPFRET pair, this fluorescent protein FRET pair improves the sensitivity of the reaction, reduces imaging phototoxicity, and has superior properties compared with other GFP-RFP pairs, and this scheme has also been successfully used in living cells Imaging of Zn 2+ aggregation and CaMKIIα activity. However, the disadvantage is that Clover has weak photostability and is prone to photobleaching under continuous light irradiation. The light intensity of mRuby2 is not high, so the use of this fluorescent protein FRET pair is limited (Lam, AJ et al. Improving FRET dynamic range with bright green and red fluorescent proteins. Nat Methods 9, 1005-1012 (2012)). The entire content of this document is hereby incorporated by reference in the present invention.
发明内容Contents of the invention
本发明主要是为了寻找高效的可用于光学成像的基因编码生物传感器的荧光蛋白。The main purpose of the present invention is to search for highly efficient fluorescent proteins that can be used for optical imaging gene-encoded biosensors.
本发明通过定点突变技术，在现有技术荧光蛋白mRuby2和mClover的基础上进行氨基酸位点突变，而得到突变的新红色荧光蛋白和绿色荧光蛋白，实现了蛋白光物理学性质的提高。The present invention uses site-directed mutation technology to carry out amino acid site mutation on the basis of fluorescent proteins mRuby2 and mClover in the prior art to obtain mutated new red fluorescent protein and green fluorescent protein, thereby realizing the improvement of protein photophysical properties.
具体而言，一方面，本发明采用定点突变技术在mRuby2基础上进行定点诱变获得了本发明的新红色荧光蛋白。本发明的该新的红色荧光蛋白的氨基酸序列与mRuby2的氨基酸序列(mRuby2的氨基酸序列可参见图1a)相比，具有以下突变位点：M160I。M160I可以让突变蛋白相比mRuby2更亮，在mRuby2基础上具有M160I突变的蛋白具有比未突变的mRuby2较高的光强度。Specifically, on the one hand, the present invention uses site-directed mutagenesis technology to perform site-directed mutagenesis on the basis of mRuby2 to obtain the new red fluorescent protein of the present invention. Compared with the amino acid sequence of mRuby2 (the amino acid sequence of mRuby2 can be seen in FIG. 1 a ), the amino acid sequence of the new red fluorescent protein of the present invention has the following mutation site: M160I. M160I can make the mutant protein brighter than mRuby2, and the protein with M160I mutation on the basis of mRuby2 has a higher light intensity than the unmutated mRuby2.
根据本发明的具体实施方案，本发明的新的红色荧光蛋白的氨基酸序列与mRuby2的氨基酸序列相比，还具有以下突变位点：N33R,M36E,T38V,K74A,G75D,M105T,C114E,H118N,Q120K,H159D,S171H,S173N,I192V,L202I,M209T,F210Y,H216V,F221Y,A222S,G223N中的一个或多个的组合。这些突变位点会让蛋白成熟或折叠变好。根据本发明的具体实施方案，本发明的红色荧光蛋白，其为选自以下(a)或(b)的蛋白：According to a specific embodiment of the present invention, compared with the amino acid sequence of mRuby2, the amino acid sequence of the new red fluorescent protein of the present invention also has the following mutation sites: N33R, M36E, T38V, K74A, G75D, M105T, C114E, H118N, A combination of one or more of Q120K, H159D, S171H, S173N, I192V, L202I, M209T, F210Y, H216V, F221Y, A222S, G223N. These mutations allow the protein to mature or fold better. According to a specific embodiment of the present invention, the red fluorescent protein of the present invention is a protein selected from the following (a) or (b):
(a)具有如SEQIDNo.2所示氨基酸序列的蛋白；(a) a protein having an amino acid sequence as shown in SEQ ID No.2;
(b)在(a)限定的氨基酸序列中经过取代、缺失或添加一个或几个氨基酸且与(a)具有相同功能的由(a)衍生的蛋白。其中，所述的“相同功能”是指相比于mRuby2蛋白光物理学性质提高(例如亮度提高)的功能。(b) A protein derived from (a) that has the same function as (a) through substitution, deletion or addition of one or several amino acids in the amino acid sequence defined in (a). Wherein, the "same function" refers to a function with improved photophysical properties (such as increased brightness) compared with mRuby2 protein.
根据本发明的一优选具体实施方案，本发明的新的红色荧光蛋白的氨基酸序列参见SEQIDNo.2所示(其中，所述的M160I突变位点对应SEQIDNo.2氨基酸序列的第164位，所述的N33R突变位点对应SEQIDNo.2氨基酸序列的第37位，……，依次类推)，本发明中命名该蛋白为mRuby3。本发明设计的优选的编码该蛋白mRuby3的基因序列参见SEQIDNo.1所示。According to a preferred embodiment of the present invention, the amino acid sequence of the new red fluorescent protein of the present invention is shown in SEQ ID No. 2 (wherein, the M160I mutation site corresponds to the 164th position of the amino acid sequence of SEQ ID No. 2, and the The N33R mutation site corresponds to the 37th position of the amino acid sequence of SEQIDNo.2, ..., and so on), and the protein is named mRuby3 in the present invention. The preferred gene sequence encoding the protein mRuby3 designed by the present invention is shown in SEQ ID No.1.
mRuby3的激发光谱和发射光谱的峰值分别在约558nm和592nm处，与mRuby2相比出现了蓝移。在峰值处的消光系数为128mM-1cm-1，量子产率为0.45，所以mRuby3的光强度比mRuby2高出35％，所以它是目前为止最亮的单体红色荧光蛋白。除此之外，mRuby3的光稳定性很好，在弧光灯的照射下，mRuby3的半衰期为349秒，长于mRuby2的123秒和TagRFP-T的337秒。在光漂白动力学方面，mRuby3表现出单指数关系，它的解离常数值为4.8，与mRuby2相比耐酸性相似。mRuby3是目前为止亮度最亮和光稳定最好的红色荧光蛋白The peaks of the excitation and emission spectra of mRuby3 are at about 558nm and 592nm, respectively, which are blue-shifted compared with mRuby2. The extinction coefficient at the peak is 128mM -1 cm -1 , and the quantum yield is 0.45, 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, the photostability of mRuby3 is very good. Under the irradiation of arc lamp, the half-life of mRuby3 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 monoexponential relationship, and its dissociation constant value was 4.8, which was similar to acid resistance compared with mRuby2. mRuby3 is the brightest and best photostable red fluorescent protein so far
另一方面，本发明还提供了mRuby3的融合蛋白，例如，所述mRuby3与mClover、mClover3、mNeonGreen或EGFP的融合蛋白。本发明通过实验证明了mRuby3的融合蛋白能够准确的与哺乳动物细胞系中的重要的亚细胞目标区域相结合。mRuby3在哺乳动物细胞系中所表达的信号强度，比红色荧光蛋白mRuby2、FusionRed和mCherry高出至少100％。mRuby3是一种比mRuby2更高效的荧光共振能量转移受体。On the other hand, the present invention also provides a fusion protein of mRuby3, for example, a fusion protein of mRuby3 and mClover, mClover3, mNeonGreen or EGFP. The present invention proves through experiments that the fusion protein of mRuby3 can accurately combine with important subcellular target regions in mammalian cell lines. The signal intensity of mRuby3 expressed in mammalian cell lines is at least 100% higher than that of red fluorescent proteins mRuby2, FusionRed and mCherry. mRuby3 is a more efficient fluorescence resonance energy transfer receptor than mRuby2.
另一方面，本发明中同样还对绿色荧光蛋白Clover进行了改造，得到光亮度和光稳定性有所提升且可以作为mRuby3荧光共振能量转移高效供体的新的绿色荧光蛋白。On the other hand, in the present invention, the green fluorescent protein Clover is also modified to obtain a new green fluorescent protein that has improved brightness and photostability and can be used as an efficient donor for mRuby3 fluorescence resonance energy transfer.
根据本发明的具体实施方案，本发明提供的新的绿色荧光蛋白的氨基酸序列与Clover的氨基酸序列相比，具有以下突变位点：N149Y。According to a specific embodiment of the present invention, compared with the amino acid sequence of Clover, the amino acid sequence of the new green fluorescent protein provided by the present invention has the following mutation site: N149Y.
根据本发明的优选具体实施方案，本发明的新的绿色荧光蛋白的氨基酸序列与Clover的氨基酸序列相比，还具有以下突变位点：G160S或G160C；According to a preferred specific embodiment of the present invention, compared with the amino acid sequence of Clover, the amino acid sequence of the new green fluorescent protein of the present invention also has the following mutation sites: G160S or G160C;
根据本发明的优选具体实施方案，本发明的新的绿色荧光蛋白的氨基酸序列与Clover的氨基酸序列相比，还具有以下突变位点：A206K。According to a preferred specific embodiment of the present invention, compared with the amino acid sequence of Clover, the amino acid sequence of the new green fluorescent protein of the present invention also has the following mutation site: A206K.
根据本发明的具体实施方案，本发明提供的新的绿色荧光蛋白，其为选自以下(a)或(b)的蛋白：According to a specific embodiment of the present invention, the novel green fluorescent protein provided by the present invention is a protein selected from the following (a) or (b):
(a)具有如SEQIDNo.4、SEQIDNo.5或SEQIDNo.6所示氨基酸序列的蛋白；(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)在(a)限定的氨基酸序列中经过取代、缺失或添加一个或几个氨基酸且与(a)具有相同功能的由(a)衍生的蛋白。其中，所述的“相同功能”是指相比于Clover蛋白光物理学性质提高(例如光稳定性提高)的功能。(b) A protein derived from (a) that has the same function as (a) through substitution, deletion or addition of one or several amino acids in the amino acid sequence defined in (a). Wherein, the "same function" refers to a function with improved photophysical properties (for example, improved photostability) compared with Clover protein.
在本发明的一具体实施方案中，本发明的新的绿色荧光蛋白，其氨基酸序列与Clover的氨基酸序列相比，仅具有以下突变位点：N149Y。本发明命名该新的绿色荧光蛋白为Clover1.5，具体地，其氨基酸序列参见SEQIDNo.5所示(其中，所述的N149Y突变位点对应SEQIDNo.5氨基酸序列的第150位)。In a specific embodiment of the present invention, compared with the amino acid sequence of Clover, the amino acid sequence of the new green fluorescent protein of the present invention only has the following mutation site: N149Y. The present invention names the new green fluorescent protein as Clover1.5, specifically, its amino acid sequence is shown in SEQ ID No. 5 (wherein, the N149Y mutation site corresponds to the 150th amino acid sequence of SEQ ID No. 5).
在本发明的一具体实施方案中，本发明的新的绿色荧光蛋白，其氨基酸序列与Clover的氨基酸序列相比，仅具有以下两个突变位点：N149Y和G160S。本发明命名该新的绿色荧光蛋白为dClover2，具体地，其氨基酸序列参见SEQIDNo.6所示(其中，所述的N149Y突变位点对应SEQIDNo.6氨基酸序列的第150位，所述的G160S突变位点对应SEQIDNo.6氨基酸序列的第161位)。In a specific embodiment of the present invention, compared with the amino acid sequence of Clover, the amino acid sequence of the new green fluorescent protein of the present invention only has the following two mutation sites: N149Y and G160S. The present invention names the new green fluorescent protein as dClover2, specifically, its amino acid sequence is shown in SEQIDNo.6 (wherein, the N149Y mutation site corresponds to the 150th amino acid sequence of SEQIDNo.6, and the G160S mutation The position corresponds to the 161st position of the amino acid sequence of SEQ ID No.6).
在本发明的一具体实施方案中，本发明的新的绿色荧光蛋白，其氨基酸序列与Clover的氨基酸序列相比，仅具有以下三个突变位点：N149Y、G160C和A206K。本发明命名该新的绿色荧光蛋白为mClover3，具体地，其氨基酸序列参见SEQIDNo.4所示(其中，所述的N149Y突变位点对应SEQIDNo.4氨基酸序列的第150位，所述的G160C突变位点对应SEQIDNo.4氨基酸序列的第161位，所述的A206K突变位点对应SEQIDNo.4氨基酸序列的第207位)，本发明设计的优选的编码该蛋白mClover3的基因序列参见SEQIDNo.3所示。In a specific embodiment of the present invention, compared with the amino acid sequence of Clover, the amino acid sequence of the new green fluorescent protein of the present invention only has the following three mutation sites: N149Y, G160C and A206K. The present invention names the new green fluorescent protein as mClover3, specifically, its amino acid sequence is shown in SEQIDNo.4 (wherein, the N149Y mutation site corresponds to the 150th amino acid sequence of SEQIDNo.4, and the G160C mutation The site corresponds to the 161st position of the amino acid sequence of SEQIDNo.4, and the A206K mutation site corresponds to the 207th position of the amino acid sequence of SEQIDNo.4), and the preferred gene sequence encoding the protein mClover3 designed by the present invention is shown in SEQIDNo.3 Show.
本发明的新的绿色荧光蛋白，在体内，dClover2在亮度归一化情况下光稳定性的半衰期为98秒，mClover3在体内时在亮度归一化情况下光稳定性的半衰期为80秒，而Clover只有50秒。For the new green fluorescent protein of the present invention, in vivo, the half-life of dClover2 is 98 seconds under normalized brightness conditions, and the half-life of mClover3 under normalized brightness conditions is 80 seconds, while mClover3 is in vivo. Clover only has 50 seconds.
另一方面，本发明还提供了所述绿色荧光蛋白的融合蛋白，例如，mClover3与mRuby3的融合蛋白。通过在哺乳动物细胞系中对mClover3系统性地进行测试，证明了mClover3的融合蛋白能够准确的与哺乳动物细胞系中的重要的亚细胞目标区域相结合，mClover3和mNeonGreen相比与mEGFP拥有更高的荧光共振能量转移效率，mClover3-mRuby3和mNeonGreen-mRuby3的效率也比Clover-mRuby3要高，mClover3和mNeonGreen是mRuby3的最有效的供体。On the other hand, the present invention also provides the fusion protein of the green fluorescent protein, for example, the fusion protein of mClover3 and mRuby3. By systematically testing mClover3 in mammalian cell lines, it is proved that the fusion protein of mClover3 can accurately bind to important subcellular target regions in mammalian cell lines, and mClover3 and mNeonGreen have a higher concentration than mEGFP The fluorescence resonance energy transfer efficiency of mClover3-mRuby3 and mNeonGreen-mRuby3 is also higher than that of Clover-mRuby3, mClover3 and mNeonGreen are the most efficient donors of mRuby3.
另一方面，本发明还提供了所述的红色荧光蛋白在作为荧光共振能量转移受体中的应用。具体地，其中，所述荧光共振能量转移的供体可选自以下蛋白：mEGFP、Envy、mNeonGreen、Clover或本发明所述的新的绿色荧光蛋白。On the other hand, the present invention also provides the application of the red fluorescent protein as a fluorescence resonance energy transfer acceptor. 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 described in the present invention.
本发明还提供了所述的绿色荧光蛋白在作为荧光共振能量转移供体中的应用。具体地，其中，所述荧光共振能量转移的受体选自以下蛋白：mCherry、mKate2、FusionRed、mRuby2或mRuby3。The invention also provides the application of the green fluorescent protein as a fluorescence resonance energy transfer donor. Specifically, wherein the receptor for fluorescence resonance energy transfer is selected from the following proteins: mCherry, mKate2, FusionRed, mRuby2 or mRuby3.
本发明还提供了一种能用于荧光共振能量转移成像的蛋白对，其包括：The present invention also provides a protein pair that can be used for fluorescence resonance energy transfer imaging, which includes:
本发明所述的新的红色荧光蛋白作为荧光共振能量转移受体；和/或The new red fluorescent protein of the present invention is used as a fluorescence resonance energy transfer acceptor; and/or
本发明所述的新的绿色荧光蛋白作为荧光共振能量转移供体。The new green fluorescent protein described in the present invention is used as a fluorescence resonance energy transfer donor.
本发明还提供了一种可用于光学成像的基因编码生物传感器，该生物传感器包括：The present invention also provides a gene-encoded biosensor that can be used for optical imaging, the biosensor comprising:
本发明所述的新的红色荧光蛋白作为荧光共振能量转移受体；和/或The new red fluorescent protein of the present invention is used as a fluorescence resonance energy transfer acceptor; and/or
本发明所述的新的绿色荧光蛋白作为荧光共振能量转移供体。The new green fluorescent protein described in the present invention is used as a fluorescence resonance energy transfer donor.
与普遍使用的基因编码的生物传感器相比，本发明的传感器在荧光成像过程中可以有效降低成像背景干扰信号，显著提高了荧光成像的灵敏度，使得成像更加清晰。由于该传感器的光稳定性有了极大地提高，有效的延长了可成像时间，使得长时间观察某种反应或现象更加容易。由于该传感器光毒性的减小，减轻了实验过程中对细胞的正常反应和功能的影响，使得实验结误差减小，实验结果更贴近真实。Compared with commonly used gene-encoded biosensors, the sensor of the present invention can effectively reduce imaging background interference signals during the fluorescence imaging process, significantly improve the sensitivity of fluorescence imaging, and make the imaging clearer. Since the photostability of the sensor has been greatly improved, the imaging time is effectively extended, making it easier to observe a certain reaction or phenomenon for a long time. Due to the reduction of the phototoxicity of the sensor, the impact on the normal response and function of the cells during the experiment is alleviated, the error of the experimental junction is reduced, and the experimental results are closer to reality.
具体地，在本发明的一具体实施方案中，分别构建了以Clover和mRuby3为FRET对的Camuiα传感器Camuiα-CR3，mClover3和mRuby3为FRET对的Camuiα传感器Camuiα-C3R3，mNeonGreen和mRuby3为FRET对的Camuiα传感器Camuiα-NR3，测试在Camuiα指示器中新的红色和绿色荧光蛋白在提高FRET基线的能力，结果显示mClover3-mRuby3和mNeonGreen3-mRuby3在Camuiα中改善了反应效果。Specifically, in a specific embodiment of the present invention, the Camuiα sensor Camuiα-CR3 with Clover and mRuby3 as the FRET pair, the Camuiα sensor Camuiα-C3R3 with mClover3 and mRuby3 as the FRET pair, and the Camuiα-C3R3 with mNeonGreen and mRuby3 as the FRET pair were respectively constructed. The Camuiα sensor, Camuiα-NR3, tested the ability of new red and green fluorescent proteins in Camuiα indicators to improve the FRET baseline, and the results showed that mClover3-mRuby3 and mNeonGreen3-mRuby3 improved the response in Camuiα.
另一方面，本发明还开发出了一套评估荧光蛋白在哺乳动物细胞系中作为独立标记和基因编码生物传感器的标准评估方法，通过该评估方法检验荧光蛋白衍生物在哺乳动物细胞系中的表达所产生的荧光信号强度。On the other hand, the present invention has also developed a set of standard evaluation methods for evaluating fluorescent proteins as independent markers and gene-encoded biosensors in mammalian cell lines, by which the detection of fluorescent protein derivatives in mammalian cell lines Express the resulting fluorescence signal intensity.
本发明与普遍使用的基因编码的生物传感器相比有5大优点：(1)选取绿色和红色荧光蛋白相融合，代替原有青色和黄色荧光蛋白相融合，以绿和红两种颜色的荧光团相搭配，使得光谱分隔的距离变大，减轻了干扰，提高了灵敏度。(2)荧光蛋白的光稳定性出现了极大的提高，荧光蛋白mClover3相比于前一代Clover提高了60％。而mRuby3相比于前一代mRuby2提高了200％，使得mRuby3蛋白成为目前为止光稳定性最强的单体红色荧光蛋白。(3)荧光蛋白的光亮度也有较大的提高，例如mRuby3比前一代mRuby2要亮35％，使得mRuby3成为目前为止光亮度最亮的红色荧光蛋白。(4)高表达，经过转染哺乳动物细胞系后，对细胞成像灵敏度高于现在普遍使用的生物传感器。(5)mRuby3是延时拍摄成像和光量子受限制情况下检测蛋白的良好探针，包括快速延时拍摄成像和单分子成像。Compared with the commonly used gene-encoded biosensors, the present invention has five major advantages: (1) select the fusion of green and red fluorescent proteins to replace the fusion of the original cyan and yellow fluorescent proteins, and use green and red fluorescent proteins The combination of clusters makes the distance of spectral separation larger, reduces interference and improves sensitivity. (2) The photostability of fluorescent proteins has been greatly improved, and the fluorescent protein mClover3 has increased by 60% compared with the previous generation Clover. Compared with the previous generation of mRuby2, mRuby3 has increased by 200%, making mRuby3 protein the most photostable monomeric red fluorescent protein so far. (3) The brightness of fluorescent proteins has also been greatly improved. For example, mRuby3 is 35% brighter than the previous generation mRuby2, making mRuby3 the brightest red fluorescent protein so far. (4) High expression, after transfection of mammalian cell lines, the sensitivity to cell imaging is higher than that of biosensors commonly used today. (5) mRuby3 is a good probe for time-lapse imaging and protein detection under the condition of photon limitation, including fast time-lapse imaging and single-molecule imaging.
附图说明Description of drawings
图1a显示mRuby3与mRuby2蛋白质一级结构的序列及对比结果。Figure 1a shows the sequence and comparison results of the primary structures of mRuby3 and mRuby2 proteins.
图1b为mRuby3与mRuby2的突变对比晶体结构图。Fig. 1b is a comparison crystal structure of mRuby3 and mRuby2 mutations.
图1c为红色荧光蛋白mCherry、mKate2、FusionRed、mRuby2和mRuby3的吸收图(左)与发射图(右)。Figure 1c shows the absorption (left) and emission (right) maps of the red fluorescent proteins mCherry, mKate2, FusionRed, mRuby2 and mRuby3.
图1d显示了mRuby3、mRuby2和mRuby2-M160I突变体在细菌培养箱中培养后的荧光图。Figure 1d shows the fluorescence profiles of mRuby3, mRuby2, and mRuby2-M160I mutants grown in a bacterial incubator.
图2a为在HeLa细胞中表达mRuby3融合蛋白后对特定的亚细胞结构的荧光成像图。从左到右：mRuby3-7aa-actin(肌动蛋白细胞骨架)，mRuby3-6aa-tubulin(微管)，connexin43(细胞粘附接点)-7aa-mRuby3，mRuby3-10aa-H2B(核小体)。中间aa代表连接两个蛋白之间的距离的单位，以单个氨基酸距离为1个aa。Fig. 2a is a fluorescence imaging image of specific subcellular structures after expressing mRuby3 fusion protein in HeLa cells. From left to right: mRuby3-7aa-actin (actin cytoskeleton), mRuby3-6aa-tubulin (microtubules), connexin43 (cell adhesion junction)-7aa-mRuby3, mRuby3-10aa-H2B (nucleosome) . The middle aa represents the unit of distance between two proteins, and the distance between a single amino acid is 1 aa.
图2b显示在HEK293A和HeLa细胞中四种单体红色荧光蛋白mCherry，FusionRed，mRuby2和mRuby3的亮度比较。Figure 2b shows the brightness comparison of four monomeric red fluorescent proteins mCherry, FusionRed, mRuby2 and mRuby3 in HEK293A and HeLa cells.
图3a显示mClover3、dClover2和Clover的蛋白质一级结构的序列及对比结果。Figure 3a shows the sequences and comparison results of the protein primary structures of mClover3, dClover2 and Clover.
图3b为mClover3与Clover相比突变位点的晶体结构图。Figure 3b is a crystal structure diagram of the mutation site of mClover3 compared with Clover.
图3c为绿色荧光蛋白mEGFP、Envy、mNeonGreen、Clover和Clover3的吸收(左)和发射(右)光谱图。Figure 3c is the absorption (left) and emission (right) spectra of green fluorescent proteins mEGFP, Envy, mNeonGreen, Clover and Clover3.
图4a为在HeLa细胞中表达mClover3融合蛋白后对特定的亚细胞结构的荧光成像图。从左到右：mClover3-7aa-actin(肌动蛋白细胞骨架)，mClover3-6aa-tubulin(微管)，connexin43(细胞粘附接点)-7aa-mClover3，mClover3-10aa-H2B(核小体)。中间aa代表连接两个蛋白之间的距离的单位，以单个氨基酸距离为1个aa。Fig. 4a is a fluorescence imaging diagram of specific subcellular structures after expressing mClover3 fusion protein in HeLa cells. From left to right: mClover3-7aa-actin (actin cytoskeleton), mClover3-6aa-tubulin (microtubules), connexin43 (cell adhesion junction)-7aa-mClover3, mClover3-10aa-H2B (nucleosomes) . The middle aa represents the unit of distance between two proteins, and the distance between a single amino acid is 1 aa.
图4b显示在HEK293A和HeLa细胞中通过表达GFP-P2A-mCherry，对六种单体绿色荧光蛋白的亮度比较。Figure 4b shows a comparison of the brightness of six monomeric green fluorescent proteins by expressing GFP-P2A-mCherry in HEK293A and HeLa cells.
图4c为在HEK293A和HeLa细胞中三种GFP-mRuby3蛋白的荧光共振能量转移的效率对比图。Fig. 4c is a graph comparing the efficiency of fluorescence resonance energy transfer of three GFP-mRuby3 proteins in HEK293A and HeLa cells.
图5a显示与Camuiα连接的绿色/红色FRET对的结构。Figure 5a shows the structure of the green/red FRET pair linked to Camuiα.
图5b显示绿色/红色表达Camuiα的HeLa细胞在没有钙离子载体刺激的情况下供体/受体的发射比率(RDA)。数据以均值±标准差形式表现。Figure 5b shows the donor/acceptor emission ratio (RDA) of green/red Camuiα-expressing HeLa cells in the absence of calcium ionophore stimulation. Data are presented as mean ± standard deviation.
图5c中，左图片为平均供体/受体的发射比率(RDA)随时间的变化图，右图片为HeLa细胞在钙离子载体刺激下反应所得到的强度比率图。每个细胞的发射比率的变化在图中用灰色线表示，平均值用黑色线表示。数据以均值±标准差形式表现。将Camuiα-CR分别与Camuiα-C3R3和Camuiα-NR3在激发峰值处的比率有统计学差异。In Fig. 5c, the left picture shows the change of average donor/acceptor emission ratio (RDA) over time, and the right picture shows the intensity ratio of HeLa cells in response to calcium ionophore stimulation. The change in the firing ratio per cell is represented in the graph by the gray line and the mean is represented by the black line. Data are presented as mean ± standard deviation. The ratios of Camuiα-CR to Camuiα-C3R3 and Camuiα-NR3 at the excitation peak were statistically different.
具体实施方式detailed description
为了更清楚地理解本发明，现参照下列实施例及附图进一步描述本发明。实施例仅用于解释而不以任何方式限制本发明。以下各实验中，各原始试剂材料均可商购获得，未注明具体条件的实验方法包括定点突变技术、融合蛋白技术、人工脂质体细胞转染技术等为所属领域熟知的常规方法和常规条件，或按照仪器制造商所建议的条件。In order to understand the present invention more clearly, the present invention will now be further described with reference to the following examples and accompanying drawings. The examples are for illustration only and do not limit the invention in any way. In the following experiments, each original reagent material can be obtained commercially, and the experimental methods without specifying specific conditions include site-directed mutagenesis technology, fusion protein technology, artificial liposome cell transfection technology, etc., which are conventional methods and conventional methods well known in the art conditions, or as recommended by the instrument manufacturer.
本发明各实验中所用主要技术和实验方案包括：Used main technique and experimental scheme in each experiment of the present invention comprise:
(1)荧光共振能量转移的距离模型。(1) The distance model of fluorescence resonance energy transfer.
建立的模型的公式为 表示在某一波长处的荧光强度。εD(λex)表示在激发波长下供体的消光系数。E表示荧光共振能量转移效率。是表示供体的量子产率。fD(λ)表示在波长λ处供体的已标准化的发射光。εA(λex)是表示在激发波长下受体的消光系数。而内部荧光基团距离(interfluorophoredistance,r)通过福斯特()公式E＝1/(1+(r6/r0 6)算出，r0为福斯特()半径。The formula of the established model is Indicates the fluorescence intensity at a certain wavelength. ε D (λ ex ) represents the extinction coefficient of the donor at the excitation wavelength. E represents the fluorescence resonance energy transfer efficiency. is the quantum yield of the donor. f D (λ) denotes the normalized emission of the donor at wavelength λ. ε A (λ ex ) represents the extinction coefficient of the acceptor at the excitation wavelength. And the interfluorophore distance (interfluorophoredistance, r) by Foster ( ) Calculated by the formula E=1/(1+(r 6 /r 0 6 ), r 0 is Foster ( )radius.
(2)重组质粒构建(2) Recombinant plasmid construction
质粒构建包括：聚合酶链式反应(PCR)，重叠延伸聚合酶链式反应(overlapPCR)，限制性酶切反应和In-Fusion连接，转化DH5α细菌。所有重组载体通过测序鉴定。Plasmid construction includes: polymerase chain reaction (PCR), overlap extension polymerase chain reaction (overlapPCR), restriction enzyme digestion reaction and In-Fusion ligation, transforming DH5α bacteria. All recombinant vectors were identified by sequencing.
(3)HeLa和HEK293A细胞培养和转染(3) HeLa and HEK293A cell culture and transfection
利用脂质体转染法将重组质粒转入HeLa和HEK293A细胞中，3-5小时后更换培养液，培养24小时后将细胞消化后，分装到8孔板中，再培养24小时。The recombinant plasmid was transferred into HeLa and HEK293A cells by lipofection method, and the culture medium was replaced after 3-5 hours. After 24 hours of culture, the cells were digested, distributed into 8-well plates, and cultured for another 24 hours.
(4)荧光显微镜观察融合蛋白(4) Observation of fusion protein by fluorescence microscope
对于mClover3-mRuby3融合蛋白，在转染到HeLa细胞中24-72小时后，利用FV1000激光共聚焦显微镜成像。对于mClover3，在488nm激发光下激发，收集500-600nm的光。对于mRuby3，在559nm处激发，收集570-670nm处的光。所得到的图像用软件ImageJ处理。For the mClover3-mRuby3 fusion protein, imaged using a FV1000 confocal microscope 24-72 hours after transfection into HeLa cells. For mClover3, excite at 488nm excitation light and collect light at 500-600nm. For mRuby3, excitation was at 559 nm and light was collected at 570-670 nm. The obtained images were processed with the software ImageJ.
(5)荧光蛋白的体外特征(5) In vitro characteristics of fluorescent proteins
由于荧光蛋白的N-末端连接有六个组氨酸，所以使用钴树脂纯化(HisPurCobaltResin)。而吸光度，激发光谱和发射光谱通过多功能酶标仪SafireII测量出来。消光系数通过变性方法(对于每个蛋白样品，准备完全一样两份溶液，蛋白溶解在PBS中。其中一份测量吸收峰，另外一份在1NNaOH作用下测量吸收峰，通过公式‘变性前/变性后*44000’计算出消光系数)测出，而量子产率则以Clover和mRuby2为参考(分别测量待测样品和参考蛋白的发射谱和吸收谱，然后分别计算发射谱面积和获得发射谱的吸收值，然后将发射谱面积除以吸收值，然后将待测样品的上述比值除以参考蛋白的比值，然后乘以参考蛋白的量子产率，即可得知待测蛋白的量子产率)。Since the fluorescent protein has six histidines attached to its N-terminus, it was purified using cobalt resin (HisPurCobaltResin). The absorbance, excitation spectrum and emission spectrum were measured by multifunctional microplate reader SafireII. The extinction coefficient is determined by the denaturation method (for each protein sample, two solutions are prepared exactly the same, and the protein is dissolved in PBS. One of them is used to measure the absorption peak, and the other is used to measure the absorption peak under the action of 1NNaOH, and the formula 'before denaturation/denaturation After *44000' to calculate the extinction coefficient), the quantum yield is measured with Clover and mRuby2 as a reference (respectively measure the emission spectrum and absorption spectrum of the sample to be tested and the reference protein, and then calculate the area of the emission spectrum and obtain the area of the emission spectrum The absorption value, and then divide the emission spectrum area by the absorption value, then divide the above ratio of the sample to be tested by the ratio of the reference protein, and then multiply by the quantum yield of the reference protein, the quantum yield of the protein to be tested can be obtained) .
在体外的光漂白实验中，以纯化的蛋白质在为样品，使用倒置荧光显微镜油镜，使用40×0.90-NAUPlanS-Apo物镜，使用X-Cite120-瓦金属卤化物灯在100％中性密度分别透过一个545/30nm的激发滤波器(对mRuby突变体而言)和一个485/30nm激发滤波器(对Clover突变体而言)。在金属氯化物光源连续照射下，每1秒采集一次图像(相机为ORCA-ERCCD)，倍数也调整为产生光子输出率为1000光子每秒。In the in vitro photobleaching experiment, the purified protein was used as a sample, using an inverted fluorescence microscope oil lens, using a 40×0.90-NAUPlanS-Apo objective lens, and using an X-Cite120-watt metal halide lamp at 100% neutral density, respectively. Pass through a 545/30 nm excitation filter (for mRuby mutants) and a 485/30 nm excitation filter (for Clover mutants). Under the continuous irradiation of the metal chloride light source, an image is collected every 1 second (the camera is ORCA-ERCCD), and the multiple is also adjusted to produce a photon output rate of 1000 photons per second.
凝胶过滤层析实验中使用复合凝胶柱(Superdex20030/100GLcolumn)。上样量为100μL，浓度为10μM。洗脱流度为0.5毫升/分钟。通过280nm处的吸收监测蛋白洗脱。A composite gel column (Superdex20030/100GLcolumn) was used in the gel filtration chromatography experiment. The loading volume was 100 μL, and the concentration was 10 μM. The elution flow rate was 0.5 ml/min. Protein elution was monitored by absorbance at 280 nm.
(6)基础荧光共振能量转移的测量(6) Measurement of basic fluorescence resonance energy transfer
GFP-RFP融合蛋白由C-端截断的avGFP衍生体或mNeonGreen通过连接序列融合了mRuby2或mRuby3的aa3-233。将融合蛋白转染到HEK293A和HeLa细胞中。转染后2天，将细胞转移到96孔板中检测荧光光谱。发射光谱范围为490-750nm，用470nm激发。GFP-RFP fusion proteins consist of C-terminally truncated avGFP derivatives or mNeonGreen fused to aa3-233 of mRuby2 or mRuby3 via a linker sequence. Fusion proteins were transfected into HEK293A and HeLa cells. Two days after transfection, the cells were transferred to a 96-well plate to detect fluorescence spectra. The emission spectrum ranges from 490-750nm with excitation at 470nm.
(7)在哺乳动物细胞系中比较突变体的光亮度(7) Comparing the brightness of mutants in mammalian cell lines
对哺乳动物细胞系中绿色荧光蛋白和mRuby的突变体进行比较时，以mCherry和mTurquoise2为表达内参，使用脂质体法转染重组质粒到HEK293A和HeLa细胞中，转染后2天，将细胞转移到底部透明的96孔板中检测荧光光谱。不同的荧光蛋白的设置参数如下：mTurquoise2-434/5nm-474/5nm,mCherry-587/20nm-610/5nm,GFP-430/20nm-480～650nm,RFP-550/10nm-570～670nm。相对亮度是以整合的绿色荧光蛋白或mRuby突变体发射光强除以mCherry或mTurquoise2的发射光强得到的。When comparing the mutants of green fluorescent protein and mRuby in mammalian cell lines, mCherry and mTurquoise2 were used as expression internal references, and the recombinant plasmid was transfected into HEK293A and HeLa cells by liposome method. Two days after transfection, the cells were Transfer to a 96-well plate with a transparent bottom to detect the fluorescence spectrum. The setting parameters of different fluorescent proteins are as follows: mTurquoise2-434/5nm-474/5nm, mCherry-587/20nm-610/5nm, GFP-430/20nm-480~650nm, RFP-550/10nm-570~670nm. Relative brightness was obtained by dividing the emission intensity of integrated GFP or mRuby mutants by the emission intensity of mCherry or mTurquoise2.
(8)Camuiα传感器改进和特征(8) Camui α sensor improvements and features
为了构建Camuiα-CR突变体，一个N-末端为NheI酶切位点和一个C-末端扩展编码的连接器(GFP和CaMKIIα之间的)被用来扩增C-末端截短的avGFP(不含‘GITHGMDELYK’序列)或mNeonGreen(不含‘GMDELYK’序列)。在任意一端的CaMKIIα区域通过PCR扩增成功后，对mRuby2或mRuby3也通过PCR方法扩增目的片段，反应同时在CaMKIIα区域引入一个N-末端扩展的连接器，在C-末端引入ApaI酶切位点，通过重叠延伸聚合酶链式反应将插入片段克隆到载体pcDNA3.1上。To construct Camuiα-CR mutants, an N-terminal NheI cleavage site and a C-terminal extension-encoded linker (between GFP and CaMKIIα) were used to amplify the C-terminal truncated avGFP (without containing 'GITHGMDELYK' sequence) or mNeonGreen (without 'GMDELYK' sequence). After the CaMKIIα region at either end is successfully amplified by PCR, the target fragment is also amplified by PCR for mRuby2 or mRuby3, and an N-terminal extended linker is introduced into the CaMKIIα region, and an ApaI restriction site is introduced into the C-terminal point, the insert was cloned into the vector pcDNA3.1 by overlap extension polymerase chain reaction.
在细胞被重组质粒转染后2天，用显微镜观察细胞。使用倒置荧光显微镜200M的冷ORCA-ERCCD摄像机和40×1.2-NAC-高度消色镜水浸模块观察，使用软件Micro-manager1.4，具体参数为：17-inch2.5-GHzCore2DuoMacBookProrunningMacOS10.6.8。连续不断的FRET和供体发射光谱成像通过以下滤光片获得：绿色荧光蛋白为激发HQ470/30nm和发射505AELPnm，FRET使用激发HQ470/30nm和发射BA575IFnm。Two days after the cells were transfected with the recombinant plasmids, the cells were observed with a microscope. Use an inverted fluorescence microscope with a 200M cold ORCA-ERCCD camera and a 40×1.2-NAC-height achromatic mirror water immersion module to observe, use the software Micro-manager1.4, and the specific parameters are: 17-inch2.5-GHzCore2DuoMacBookProrunningMacOS10.6.8. Sequential FRET and donor emission spectral imaging were obtained with the following filters: Excitation HQ470/30nm and emission 505AELPnm for GFP, FRET using excitation HQ470/30nm and emission BA575IFnm.
(9)比率型图像分析(9) Ratio image analysis
FRET的测量通过软件ImageJ量化。原始文件为16-位TIFF文件，以随机的方法来选择视野，转染发出荧光细胞为阳性，以未转染的细胞为背景检测。背景去掉供体的发射强度除以背景去掉FRET的强度得到的值为发射比率。使用一个全光谱查阅表(最小值为蓝色，最大值为红色)，通过受体通道产生强度调节显示。The measurement of FRET was quantified by the software ImageJ. The original file is a 16-bit TIFF file, and the field of view is randomly selected. The transfected fluorescent cells are positive, and the untransfected cells are used as the background detection. The emission intensity of the background-subtracted donor divided by the intensity of the background-subtracted FRET is the emission ratio. Using a full spectral look-up table (minimum in blue, maximum in red), an intensity-modulated display is generated through the receptor channel.
(10)统计方法(10) Statistical methods
采用方差分析和邓尼特法后验检测确定细胞中亮度测量的差异。采用T检验确定Camuiα突变体的峰值发射比率改变有没有统计学差异。作图软件为Excel和Prism。Differences in brightness measurements in cells were determined using ANOVA with Dunnett's posterior test. A t-test was used to determine whether there was a statistically significant change in the peak firing ratio of the Camuiα mutants. The graphing software is Excel and Prism.
实施例1：可用于高灵敏FRET成像的荧光蛋白对Example 1: Fluorescent protein pairs that can be used for highly sensitive FRET imaging
1新红色荧光蛋白mRuby3的性质和成像效果1 Properties and imaging effects of the new red fluorescent protein mRuby3
1.1mRuby3的结构和光物理性质1.1 Structure and photophysical properties of mRuby3
采用定点突变技术在mRuby2基础上进行定点诱变获得本发明的新红色荧光蛋白mRuby3。mRuby3的氨基酸序列参见SEQIDNo.2所示，本发明设计的优选的编码该蛋白mRuby3的基因序列参见SEQIDNo.1所示(可通过基于PCR聚合的基因合成方法获得mRuby3的DNA序列，即设计很多PCR引物，每两个引物存在18～25bp的重叠，然后通过overlapPCR的方法将所有引物聚合，从而行程一个完整的基因)。The novel red fluorescent protein mRuby3 of the present invention is obtained by performing site-directed mutagenesis on the basis of mRuby2 by site-directed mutagenesis technology. The amino acid sequence of mRuby3 is shown in SEQIDNo.2, and the gene sequence of the preferred encoding protein mRuby3 of the present invention is shown in SEQIDNo.1 (the DNA sequence of mRuby3 can be obtained by the gene synthesis method based on PCR polymerization, that is, many PCRs are designed. Primers, every two primers have an overlap of 18-25 bp, and then all the primers are aggregated by the method of overlapPCR, so as to travel a complete gene).
mRuby3与上一代的mRuby2相比氨基酸序列有21个替换，具体为：N33R,M36E,T38V,K74A,G75D,M105T,C114E,H118N,Q120K,H159D,M160I,S171H,S173N,I192V,L202I,M209T,F210Y,H216V,F221Y,A222S,G223N，可参见图1a，而mRuby的晶体结构(PDB数据库ID为3U0M)可参见图1b。图1a中，形成生色团的氨基酸已经用黑色方框标出；位于蛋白外壁的突变用蓝色标出，包括N33R,T38V,M105T,C114E,H118N,Q120K,H159D,S171H,S173N,L202I,F210Y,H216V；位于内壁的突变用绿色标出，包括：M160I；位于环处的突变用橙色标出，包括：M36E,K74A,G75D,I192V,M209T,F221Y,A222S,G223N。Compared with the previous generation of mRuby2, mRuby3 has 21 substitutions in amino acid sequence, specifically: N33R, M36E, T38V, K74A, G75D, M105T, C114E, H118N, Q120K, H159D, M160I, S171H, S173N, I192V, L202I, M209T, F210Y, H216V, F221Y, A222S, G223N, see Figure 1a, and the crystal structure of mRuby (PDB database ID is 3U0M) can be seen in Figure 1b. In Figure 1a, amino acids forming chromophores have been marked with black boxes; mutations located on the outer wall of the protein are marked in blue, including N33R, T38V, M105T, C114E, H118N, Q120K, H159D, S171H, S173N, L202I, F210Y, H216V; mutations located in the inner wall are marked in green, including: M160I; mutations located in the ring are marked in orange, including: M36E, K74A, G75D, I192V, M209T, F221Y, A222S, G223N.
红色荧光蛋白mCherry、mKate2、FusionRed、mRuby2、和mRuby3的吸收图可参见图1c中左图，各蛋白发射图可参见图1c中右图。图中可见，mRuby3的激发光谱和发射光谱的峰值分别在558nm和592nm处，与mRuby2相比出现了蓝移。在峰值处的消光系数为128mM-1cm-1，量子产率为0.45(见表1)，所以mRuby3的光强度比mRuby2高出35％，所以它是目前为止最亮的单体红色荧光蛋白。除此之外，mRuby3的光稳定性很好，在弧光灯的照射下，mRuby3的半衰期为349秒，长于mRuby2的123秒和TagRFP-T的337秒。在光漂白动力学方面，mRuby3表现出单指数关系，它的解离常数值为4.8，与mRuby2相比耐酸性相似。所以mRuby3是目前为止亮度最亮和光稳定最好的红色荧光蛋白。The absorption diagrams of the red fluorescent proteins mCherry, mKate2, FusionRed, mRuby2, and mRuby3 can be found in the left panel in Figure 1c, and the emission profiles of each protein can be found in the right panel in Figure 1c. It can be seen from the figure that the peaks of the excitation spectrum and emission spectrum of mRuby3 are at 558nm and 592nm, respectively, which are blue-shifted compared with mRuby2. The extinction coefficient at the peak is 128mM -1 cm -1 , and the quantum yield is 0.45 (see Table 1), so the light intensity of mRuby3 is 35% higher than that of mRuby2, so it is by far the brightest monomeric red fluorescent protein . In addition, the photostability of mRuby3 is very good. Under the irradiation of arc lamp, the half-life of mRuby3 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 monoexponential relationship, and its dissociation constant value was 4.8, which was similar to acid resistance compared with mRuby2. So mRuby3 is the red fluorescent protein with the brightest brightness and the best photostability so far.
表1：单体绿色和红色荧光蛋白的光物理性质Table 1: Photophysical properties of monomeric green and red fluorescent proteins
表注：b为激发光峰值时所在的波长。c为发射光峰值时所在的波长。d为最大消光系数。e为荧光量子产率。f为蛋白光亮度的相对值。g为解离常数。h为蛋白的光稳定性，表示在弧光灯照射下每个分子每秒从1000个光子光漂白为500个光子所用的时间，时间单位为秒。Note: b is the wavelength of the excitation light peak. 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 protein brightness. g is the dissociation constant. h is the photostability of the protein, which represents the time it takes for each molecule to photobleach from 1000 photons to 500 photons per second under the irradiation of an arc lamp, and the time unit is second.
mRuby3与mRuby2相比的各突变位点中，M160I让蛋白更亮，但蛋白成熟或折叠变差；而其它位点则让蛋白成熟或折叠变好。在本发明中，还在mRuby2的基础上定点突变得到了与mRuby2相比仅具有M160I突变位点的突变体，本发明中命名为mRuby2-M160I。图1d中的图片(1)显示了mRuby2(右边)和mRuby2-M160I(左边)突变体在细菌培养箱中过夜培养24h的荧光图，图1d中的图片(2)显示了mRuby2(右边)和mRuby2-M160I(左边)突变体在细菌培养箱中培养48h的荧光图。从图中可知，mRuby2-M160I由于成熟或折叠比较慢，因此24h培养后比mRuby2要暗一些，但随着培养时间的延长，最终要比mRuby2要亮。图1d中的图片(3)为mRuby3和mRuby2在细菌培养箱中过夜培养24h后的荧光图(图中左边为mRuby3，右边为mRuby2)。Among the mutation sites in mRuby3 compared with mRuby2, M160I makes the protein brighter, but the protein matures or folds poorly; while other sites make the protein mature or fold better. In the present invention, the site-directed mutation on the basis of mRuby2 is also used to obtain a mutant with only the M160I mutation site compared with mRuby2, which is named mRuby2-M160I in the present invention. Picture (1) in Fig. 1d shows the fluorescence images of mRuby2 (right) and mRuby2-M160I (left) mutants cultured overnight in a bacterial incubator for 24h, and picture (2) in Fig. 1d shows mRuby2 (right) and The fluorescence image of the mRuby2-M160I (left) mutant cultured in the bacterial incubator for 48 hours. It can be seen from the figure that mRuby2-M160I is darker than mRuby2 after 24 hours of culture due to slow maturation or folding, but eventually becomes brighter than mRuby2 as the culture time prolongs. Picture (3) in Fig. 1d is the fluorescence picture of mRuby3 and mRuby2 after 24 hours of overnight culture in the bacterial incubator (mRuby3 is on the left in the figure, mRuby2 is on the right).
1.2mRuby3在哺乳动物细胞系中成像1.2 Imaging of mRuby3 in mammalian cell lines
在哺乳动物细胞系中比较突变体的光亮度。图2a为在HeLa细胞中表达mRuby3融合蛋白后对特定的亚细胞结构的荧光成像图。从左到右：mRuby3-7aa-actin(肌动蛋白细胞骨架)，mRuby3-6aa-tubulin(微管)，connexin43(细胞粘附接点)-7aa-mRuby3，mRuby3-10aa-H2B(核小体)。中间aa代表连接两个蛋白之间的距离的单位，以单个氨基酸距离为1个aa。证明了mRuby3的融合蛋白(PCR扩增获得mRuby3序列，然后将此连接入EcoRI和BglII双酶切的pEGFP-C1载体中构建pmRuby3-C1，然后分别扩增上诉亚细胞定位序列，然后通过in-fusion技术连入BamHI和EcoRI双酶切的pmRuby3-C1载体中构建融合蛋白)能够准确的与哺乳动物细胞系中的重要的亚细胞目标区域相结合。Compare the brightness of mutants in mammalian cell lines. Fig. 2a is a fluorescence imaging image of specific subcellular structures after expressing mRuby3 fusion protein in HeLa cells. From left to right: mRuby3-7aa-actin (actin cytoskeleton), mRuby3-6aa-tubulin (microtubules), connexin43 (cell adhesion junction)-7aa-mRuby3, mRuby3-10aa-H2B (nucleosome) . The middle aa represents the unit of distance between two proteins, and the distance between a single amino acid is 1 aa. The fusion protein of mRuby3 is proved (PCR amplification obtains the mRuby3 sequence, and then connects this into the pEGFP-C1 carrier of EcoRI and BglII double digestion to construct pmRuby3-C1, then amplifies the subcellular localization sequence respectively, and then passes in- The fusion technology is connected to the pmRuby3-C1 vector cut by BamHI and EcoRI to construct the fusion protein) that can accurately combine with important subcellular target regions in mammalian cell lines.
然后将mRuby3在哺乳细胞内所产生的荧光信号与其它的单体的红色荧光蛋白进行比较。图2b显示了在HEK293A和HeLa细胞中四种单体红色荧光蛋白mCherry、FusionRed、mRuby2和mRuby3的亮度比较，结果显示mRuby3在HEK293A和HeLa细胞中所表达的信号强度最强，比红色荧光蛋白mRuby2、FusionRed和mCherry高出至少100％(图2b)。本发明的这种检测方法得到的结果与单独每个分子光亮度检测方法相比灵敏度更高。The fluorescent signal generated by mRuby3 in mammalian cells was then compared to other monomeric red fluorescent proteins. Figure 2b shows the brightness comparison of the four monomeric red fluorescent proteins mCherry, FusionRed, mRuby2 and mRuby3 in HEK293A and HeLa cells. The results show that the signal intensity of mRuby3 expressed in HEK293A and HeLa cells is the strongest, which is higher than that of red fluorescent protein mRuby2 , FusionRed and mCherry were at least 100% higher (Fig. 2b). The result obtained by the detection method of the present invention is more sensitive than the detection method of the brightness of each molecule alone.
由此可见，mRuby3理论上是一个FRET的优秀受体。由于mRuby2已经证明是一个高效的荧光共振能量转移受体。所以将mRuby3与mRuby2进行比较，以相同的绿色荧光蛋白Clover为供体，发现mRuby3比mRuby2更高效。It can be seen that mRuby3 is theoretically an excellent receptor for FRET. Since mRuby2 has been proved to be an efficient fluorescence resonance energy transfer acceptor. So comparing mRuby3 with mRuby2, using the same green fluorescent protein Clover as a donor, it was found that mRuby3 is more efficient than mRuby2.
2新绿色荧光蛋白mClover3的性质和成像效果2 Properties and imaging effects of the new green fluorescent protein mClover3
2.1mClover3的结构和光物理性质2.1 Structure and photophysical properties of mClover3
本发明中同样还对绿色荧光蛋白Clover进行了改造，意图得到一个光亮度和光稳定性有所提升且可以作为mRuby3荧光共振能量转移高效供体的蛋白。通过随机突变技术(采用Clontech公司的GeneMorphIIRandomMutagenesis试剂盒进行，保证1～3bp突变/1000bp)得到Clover的突变体，然后通过蓝色发光二极管的照射筛选光稳定性强的突变体。经过筛选得到Clover1.5(第149位由天冬酰胺变成酪氨酸)，dClover2(第149位由天冬氨酸变成酪氨酸，第160位由甘氨酸变成丝氨酸)。在体内，dClover2在亮度归一化情况下光稳定性的半衰期为98秒，而Clover只有50秒。消光系数为123mM–1cm–1，也比Clover的111mM–1cm–1有所提高。相对量子产率为0.8比Clover的0.76稍高。在dClover2的基础上进一步经过将第206位点处丙氨酸变为赖氨酸和将160位点处丝氨酸变半胱氨酸后，得到了单体的荧光蛋白mClover3。mClover3的氨基酸序列参见SEQIDNo.4所示，本发明设计的优选的编码该蛋白mClover3的基因序列参见SEQIDNo.3所示(可通过基因合成的方法获得mClover3的DNA序列)。In the present invention, the green fluorescent protein Clover is also modified in order to obtain a protein with improved brightness and photostability, which can be used as an efficient donor of mRuby3 fluorescence resonance energy transfer. Clover mutants were obtained by random mutation technique (GeneMorphII Random Mutagenesis Kit from Clontech Company, guaranteeing 1-3 bp mutation/1000 bp), and then mutants with strong photostability were screened by irradiation of blue light-emitting diodes. Clover1.5 (change from asparagine to tyrosine at position 149) and dClover2 (change from aspartic acid to tyrosine at position 149 and change from glycine to serine at position 160) were obtained after screening. In vivo, dClover2 has a photostable half-life of 98 seconds normalized for brightness compared to Clover's 50 seconds. The extinction coefficient of 123mM –1 cm –1 is also improved from Clover’s 111mM –1 cm –1 . The relative quantum yield of 0.8 is slightly higher than Clover's 0.76. On the basis of dClover2, after changing alanine at position 206 to lysine and serine at position 160 to cysteine, the monomeric fluorescent protein mClover3 was obtained. The amino acid sequence of mClover3 is shown in SEQ ID No. 4, and the preferred gene sequence encoding the protein mClover3 designed by the present invention is shown in SEQ ID No. 3 (the DNA sequence of mClover3 can be obtained by gene synthesis).
图3a显示了mClover3、dClover2和Clover的蛋白质一级结构的序列及对比结果。形成生色团的氨基酸已经用黑色方框标出。位于蛋白壁上的三处突变(N149,G160,A206)用橙色标出。图3b显示了mClover3与Clover相比突变位点的晶体结构。图3c显示了绿色荧光蛋白mEGFP、Envy、mNeonGreen、Clover和Clover3的吸收光谱(左图)和发射光谱(右图)，可以看出mClover3与Clover相比拥有相似的光谱，并且在亮度上也差不多。另mClover3在体内时在亮度归一化情况下光稳定性的半衰期为80秒。Figure 3a shows the sequence and comparison results of the protein primary structures of mClover3, dClover2 and Clover. Amino acids that form chromophores have been marked with black boxes. Three mutations (N149, G160, A206) located on the protein wall are highlighted in orange. Figure 3b shows the crystal structure of the mutation site in mClover3 compared to Clover. Figure 3c shows the absorption spectra (left) and emission spectra (right) of the green fluorescent proteins mEGFP, Envy, mNeonGreen, Clover, and Clover3. It can be seen that mClover3 has a similar spectrum to Clover, and the brightness is similar . In addition, when mClover3 is in vivo, the half-life of photostability is 80 seconds under normalized brightness conditions.
2.2mClover3在哺乳动物细胞系中成像2.2 mClover3 Imaging in Mammalian Cell Lines
在哺乳动物细胞系中对mClover3系统性地进行了测试。mClover3 was systematically tested in mammalian cell lines.
图4a显示了在HeLa细胞中表达mClover3融合蛋白后对特定的亚细胞结构的荧光成像图。从左到右：mClover3-7aa-actin(肌动蛋白细胞骨架)，mClover3-6aa-tubulin(微管)，connexin43(细胞粘附接点)-7aa-mClover3，mClover3-10aa-H2B(核小体)。中间aa代表连接两个蛋白之间的距离的单位，以单个氨基酸距离为1个aa。证明了mRuby3的融合蛋白能够准确的与哺乳动物细胞系中的重要的亚细胞目标区域相结合。然后将mRuby和mRuby3在哺乳细胞内所产生的荧光信号与其它的单体的绿色荧光蛋白进行比较，如mEGFP、sfGFP和Envy。mEGFP是现在用途最广泛的荧光蛋白。sfGFP是mEGFP的具有高折叠效率的衍生体。而Envy在出芽酵母中被认为是亮度最大的。但是，这三种蛋白在体外提取物中都没有Clover和mClover3亮度大(表1)。但是在哺乳动物细胞系中表现出相似的亮度。而最近报道的单体mNeonGreen在体外相比mClover3要亮10％(表1)，二者有着相似的激发和发射光谱，与绿色荧光蛋白(GFP)相比出现了少许的红移，可能是荧光生色团出现了阳离子-π相互作用。Figure 4a shows the fluorescence imaging of specific subcellular structures after expressing mClover3 fusion protein in HeLa cells. From left to right: mClover3-7aa-actin (actin cytoskeleton), mClover3-6aa-tubulin (microtubules), connexin43 (cell adhesion junction)-7aa-mClover3, mClover3-10aa-H2B (nucleosomes) . The middle aa represents the unit of distance between two proteins, and the distance between a single amino acid is 1 aa. It was demonstrated that mRuby3 fusion proteins can accurately bind to important subcellular target regions in mammalian cell lines. The fluorescent signals generated by mRuby and mRuby3 in mammalian cells were then compared to those of other monomeric GFPs, such as mEGFP, sfGFP, and Envy. mEGFP is currently the most widely used fluorescent protein. sfGFP is a derivative of mEGFP with high folding efficiency. And Envy is considered the brightest among budding yeasts. However, none of these three proteins was as bright as Clover and mClover3 in in vitro extracts (Table 1). But a similar brightness was shown in mammalian cell lines. The recently reported monomer mNeonGreen is 10% brighter than mClover3 in vitro (Table 1). The two have similar excitation and emission spectra, and there is a slight red shift compared with green fluorescent protein (GFP), which may be due to fluorescence. The chromophore exhibits cation-π interactions.
在HEK293A和HeLa细胞中通过表达GFP-P2A-mCherry，比较了六种单体绿色荧光蛋白的亮度，结果参见图4b。发现在HEK293A细胞中，转染了mNeonGreen的细胞平均荧光强度是最亮的，其次是Clover或mClover3，再次是Envy，最弱的是sfGFP。mNeonGreen和mClover3、mClover3和EGFP或sfGFP之间的差异具有统计学意义(p<0.05)。在HeLa细胞中，mNeonGreen、Clover或mClover3表达后都出现很强的荧光，相互之间亮度差别不大，而Envy的亮度次之，然后是EGFP，最后是sfGFP。mClover3、Envy、EGFP和sfGFP之间的亮度差异有统计学意义。By expressing GFP-P2A-mCherry in HEK293A and HeLa cells, the brightness of six monomeric green fluorescent proteins was compared, and the results are shown in Figure 4b. It was found that in HEK293A cells, the average fluorescence intensity of cells transfected with mNeonGreen was the brightest, followed by Clover or mClover3, followed by Envy, and the weakest was sfGFP. The difference between mNeonGreen and mClover3, mClover3 and EGFP or sfGFP was statistically significant (p<0.05). In HeLa cells, mNeonGreen, Clover or mClover3 all showed strong fluorescence after expression, and there was little difference in brightness among them, while the brightness of Envy was next, then EGFP, and finally sfGFP. The difference in brightness among mClover3, Envy, EGFP and sfGFP was statistically significant.
为了寻找最适合的可以做mRuby3的荧光共振能量转移最佳供体的单体绿色荧光蛋白，将要检测的单体的绿色荧光蛋白mClover、mClover3、mNeonGreen和EGFP与mRuby3做成融合蛋白，然后在哺乳动物细胞中检测各种融合蛋白的荧光共振能量转移的效率，结果见图4c，显示实验所得的发射光光谱(红色线)与线性拟合的发射光光谱(黑色线)十分的吻合，mClover3和mNeonGreen相比与mEGFP拥有更高的荧光共振能量转移效率。mClover3-mRuby3和mNeonGreen-mRuby3的效率也比Clover-mRuby3要高(图4c)。由此可见，mClover3和mNeonGreen是mRuby3的最有效的供体。In order to find the most suitable monomeric green fluorescent protein that can be the best donor for fluorescence resonance energy transfer of mRuby3, the monomeric green fluorescent proteins mClover, mClover3, mNeonGreen and EGFP to be detected are made into fusion proteins with mRuby3, and then in breastfeeding The efficiency of fluorescence resonance energy transfer of various fusion proteins was detected in animal cells. The results are shown in Figure 4c, which shows that the emission spectrum obtained from the experiment (red line) is very consistent with the linear fitting emission spectrum (black line). mClover3 and Compared with mEGFP, mNeonGreen has higher fluorescence resonance energy transfer efficiency. mClover3-mRuby3 and mNeonGreen-mRuby3 were also more efficient than Clover-mRuby3 (Fig. 4c). Thus, mClover3 and mNeonGreen are the most effective donors of mRuby3.
3Camuiα传感器3Camuiα sensor
首先分别构建以Clover和mRuby3为FRET对的Camuiα-CR3，mClover3和mRuby3为FRET对的Camuiα-C3R3，mNeonGreen和mRuby3为FRET对的Camuiα-NR3。Firstly, Camuiα-CR3 with Clover and mRuby3 as FRET pair, Camuiα-C3R3 with mClover3 and mRuby3 as FRET pair, and Camuiα-NR3 with mNeonGreen and mRuby3 as FRET pair were respectively constructed.
图5a显示与Camuiα连接的绿色/红色FRET对的结构。实验证明在检测CaMKIIα的活性时，Clover3-mRuby3提高了荧光共振能量转移的灵敏度。在Camuiα实验中，荧光蛋白与CaMKIIα多肽的终端相融合形成FRET对。与结构分析相一致，Camuiα在不活跃状态下FRET能力高，在活化状态下FRET能力低(图5a)。Figure 5a shows the structure of the green/red FRET pair linked to Camuiα. Experiments show that Clover3-mRuby3 improves the sensitivity of fluorescence resonance energy transfer when detecting the activity of CaMKIIα. In the Camuiα assay, a fluorescent protein is fused to the termini of the CaMKIIα polypeptide to form a FRET pair. Consistent with the structural analysis, Camuiα has a high FRET capacity in the inactive state and a low FRET capacity in the activated state (Fig. 5a).
然后测试在Camuiα指示器中新的红色和绿色荧光蛋白在提高FRET基线的能力。图5b显示了绿色/红色表达Camuiα的HeLa细胞在没有钙离子载体刺激的情况下供体/受体的发射比率(RDA)，显示含有mRuby3的Camuiα-CR3的FRET基线比含有mRuby2的Camuiα-CR高(图5b)。仅仅将Clover替换为mClover3或mNeonGreen不能明显的改变基本的FRET水平(图5b)。数据以均值±标准差形式表现。The novel red and green fluorescent proteins were then tested for their ability to increase the FRET baseline in Camui alpha indicators. Figure 5b shows the donor/acceptor emission ratio (RDA) of green/red Camuiα-expressing HeLa cells in the absence of calcium ionophore stimulation, showing that the FRET baseline of Camuiα-CR3 containing mRuby3 is higher than that of Camuiα-CR containing mRuby2 high (Fig. 5b). Merely replacing Clover with mClover3 or mNeonGreen did not significantly alter basal FRET levels (Fig. 5b). Data are presented as mean ± standard deviation.
最后检测Camuiα本身效果是否会因为新的FRET对的出现有所提升。在钙离子载体的刺激下，在HeLa细胞中比较Camuiα-CR、-CR3、-C3R3和-NR3的反应。图5c的左图为平均供体/受体的发射比率(RDA)随时间的变化图，右图为HeLa细胞在钙离子载体刺激下反应所得到的强度比率图。每个细胞的发射比率的变化在图中用灰色线表示，平均值用黑色线表示。数据以均值±标准差形式表现。将Camuiα-CR分别与Camuiα-C3R3和Camuiα-NR3在激发峰值处的比率有统计学差异。Camuiα-CR3的平均反应与Camuiα-CR相似，大约在45％(图5c)。如果将mClover3和mNeonGreen取代Clover将会显著地提高Camuiα动态范围，钙诱导的增强提高了绿色/红色发射比率。在Camuiα-CR3中提高了45％，在Camuiα-C3R3中提高了70％，在Camuiα-NR3中提高了56％(图5c)。虽然Camuiα-CR3、-C3R3和-NR3拥有相似的r0值和FRET基线，但是Camuiα的敏感性出现了显著的提升。如果mNeonGreen或mClover3出现了149位由N变成Y和第206位由A变成K两个位置突变，会促进Camuiα-C3R3和Camuiα-NR3向活化状态转变。Finally, it is tested whether the effect of Camuiα itself will be improved due to the emergence of new FRET pairs. The responses of Camuiα-CR, -CR3, -C3R3 and -NR3 were compared in HeLa cells under calcium ionophore stimulation. The left panel of Fig. 5c is a plot of the average donor/acceptor emission ratio (RDA) over time, and the right panel is a plot of the intensity ratio of HeLa cells in response to calcium ionophore stimulation. The change in the firing ratio per cell is represented in the graph by the gray line and the mean is represented by the black line. Data are presented as mean ± standard deviation. The ratios of Camuiα-CR to Camuiα-C3R3 and Camuiα-NR3 at the excitation peak were statistically different. The average response of Camuiα-CR3 was similar to that of Camuiα-CR at about 45% (Fig. 5c). Replacing Clover with mClover3 and mNeonGreen will significantly increase the Camuiα dynamic range, and the calcium-induced enhancement increases the green/red emission ratio. It was increased by 45% in Camuiα-CR3, 70% in Camuiα-C3R3 and 56% in Camuiα-NR3 (Fig. 5c). Although Camuiα-CR3, -C3R3, and -NR3 had similar r 0 values and FRET baselines, Camuiα showed a significant improvement in sensitivity. If mNeonGreen or mClover3 has two mutations at position 149 from N to Y and at position 206 from A to K, it will promote the transition of Camuiα-C3R3 and Camuiα-NR3 to the active state.
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