Dark fluorescent protein darkmRuby, nucleic acid sequence, vector and application

Abstract

The application discloses a dark fluorescent protein darkmRuby, a nucleic acid sequence, a vector and application. The amino acid sequence of the dark fluorescent protein darkmRuby is shown as SEQ ID NO:1, the excitation peak is 556 nm, and the dark fluorescent protein is the first dark fluorescent protein in the 550-570 light area. The methionine at the 94th position and the phenylalanine at the 96th position of the dark fluorescent protein darkmRuby play a key role in the brightness of the dark fluorescent protein darkmRuby. The phenylalanine at the 174th position of the dark fluorescent protein darkmRuby is mutated into histidine, and the brightness of the dark fluorescent protein darkmRuby can also be significantly increased.

Description

TECHNICAL FIELD

[0001] The present application relates to biomedical optics and molecular imaging, in particular to a dark fluorescent protein darkmRuby, nucleic acid sequence, vector and application. BACKGROUND

[0002] The technical background involved in the present application includes the following aspects:

[0003] (1) Site-directed mutagenesis technology

[0004] Site-directed mutagenesis refers to introducing desired changes (usually changes in a favorable direction) into a target DNA fragment (which can be a genome or a plasmid) through methods such as polymerase chain reaction (PCR), including addition, deletion, point mutation, etc. of bases. Site-directed mutagenesis can rapidly and efficiently improve the traits and characteristics of the target protein expressed by DNA, and is a very useful means in gene research work. In vitro site-directed mutagenesis technology is an important experimental means in current research in various fields of biology and medicine, and is a powerful tool for studying the complex relationship between protein structure and function.

[0005] The structure of a protein determines its function, and the relationship between the two is one of the focuses of proteomics research. Site-directed alteration, deletion or insertion of a specific base of a known gene can change the corresponding amino acid sequence and protein structure, and studying the expression product of the mutated gene helps humans understand the relationship between protein structure and function, and explore the structure / domain of the protein. The potential application fields of site-directed mutagenesis technology are very wide, such as studying the structure of protein interaction sites, modifying the different activity or kinetic characteristics of enzymes, modifying promoters or DNA acting elements, introducing new enzyme cutting sites, improving the antigenicity or stability of proteins, studying the crystal structure of proteins, and drug development, gene therapy, etc.

[0006] (2) Fusion protein technology

[0007] Fusion protein is the expression product of two or more genes recombined through DNA recombination technology. It is a purposeful gene fusion and protein expression method for obtaining a large amount of standard fusion protein. With fusion protein technology, new proteins with multiple functions can be constructed and expressed. With further understanding of protein structure and function, and the interaction between protein molecules, the application of fusion protein technology is also becoming more and more widespread.

[0008] The basic principle of constructing a fusion protein is to delete the stop codon of the first protein, and then add the second, third, fourth… protein gene, and add a stop codon to the last gene to achieve the co-expression of multiple genes. The specific steps include:

[0009] a) Cloning of the target gene: according to the principle of gene sequence complementarity, design appropriate primer sequences, and use PCR technology to amplify different target gene fragments.

[0010] b) Recombination of multiple genes can be divided into two methods: one is recombination in a vector, two DNA fragments are cut by restriction endonuclease and recovered, and then the two gene fragments with the same endonuclease site are connected in vitro by ligase; the second method is to design overlap primers to fuse two or more genes by overlap PCR, and the second method is currently the commonly used method.

[0011] c) Cloning of the recombinant gene into a high-expression plasmid vector to construct a recombinant plasmid.

[0012] d) Transfection of the recombinant expression vector into host cells and screening and sequencing using a selection marker.

[0013] e) Induced expression of the fusion gene.

[0014] The realization of the fusion protein technology requires the construction of an expression system, and the present application constructs a prokaryotic expression system. The prokaryotic expression system uses E. coli host to express the fusion gene, the product can be secreted, the purification is simple and the cost is low. The target fusion protein can be amplified and expressed on a large scale at a low price.

[0015] (3) Fluorescent protein engineering

[0016] Fluorescent protein engineering utilizes site-directed mutagenesis to modify fluorescent protein genes: the first wild-type green fluorescent protein (wtGFP) was found in the green fluorescent protein of Aequorea victoria (avGFP), which consists of 238 amino acids and has a molecular weight of about 27 kDa. Ser65-Tyr66-Gly67 in the fluorescent protein molecule spontaneously forms a luminescent group in the presence of oxygen, which is located in the middle and surrounded by 11 β-folds to form a barrel structure, and can emit weak green fluorescence under ultraviolet excitation [Zimmer M. Green fluorescent protein (GFP) applications, structure, and related photophysical behavior. Chem Rev, 2002, 3: 759-782.]. Red fluorescent protein is derived from mushroom coral and sea anemone, and its molecular structure is similar to GFP, also forming a barrel structure by surrounding the luminescent group in the middle with β-folds. The earliest discovered red fluorescent protein DsRed and eqFP are tetramers, which are prone to aggregation in cells when used to construct more complex molecular probes or as protein tags, affecting function, and can only be used for simple cell or tissue labeling. Subsequent fluorescent protein engineering designs the central luminescent group and surrounding amino acids to modify DsRed and eqFP as templates, and develops a series of monomeric red fluorescent proteins: mRFP1, mCherry, mApple, mRuby, mKate, FusionRed, mNeptune, mCardinal and TagRFP, etc. [Shen Y, Dana H, Abdelfattah AS, Patel R, Shea J, Molina Rs, Rawal B, Rancic V, Chang YF, Wu L, Chen Y, Qian Y, Wiens MD, Hambleton N, Ballanyi K, Hughes TE, Drobizhev M, Kim DS, Koyama M, Schreiter ER, Campbell RE. A genetically encoded Ca 2+ indicator based on circularly permutated sea anemone redfluorescentprotein eqFP578. BMC Biol, 2018, 16: 9.].

[0017] (4) Dark fluorescent protein

[0018] Molecular brightness is the most important property of fluorescent proteins, which is generally calculated as the product of the extinction coefficient (EC) and the quantum yield (QY). It is generally believed that the higher the molecular brightness of a fluorescent protein, the better its performance. As a type of fluorescent protein with special functions and uses, dark fluorescent proteins generally have high extinction coefficients and very low quantum yields (<0.1), and can be used in fluorescence resonance energy transfer technology based on fluorescence lifetime imaging (FLIM-FRET) [Bryce T. Bajar, Emily S. Wang, Shu Zhang, Michael Z. Lin, Jun Chu. A Guide to Fluorescent Protein FRET Pairs. Sensors (Basel), 2018, 16(9): 1488.]. Existing dark fluorescent proteins include ShadowG [Murakoshi H, S. A., Nakahata Y, Nabekura J, A dark green fluorescent protein as an acceptor for measurement of resonance energy transfer. Sci Rep, 2015. 5.], ShadowY [Murakoshi H, S. A., Nakahata Y, Nabekura J, ShadowY: a dark yellow fluorescent protein for FLIM-based FRET measurement. Sci Rep, 2017. 7(1).], sREACh [Murakoshi H, L. S., Ryohei Yasuda R, Highly sensitive and quantitative FRET-FLIM imaging in single dendritic spines using improved non-radiative YFP. Brain Cell Biol, 2008. 36(1-4): p. 31-42.], E2-Crimson NF [Laufer J, J. A., Pule M, Beard P, In vitro characterization of genetically expressed absorbing proteins using photoacoustic spectroscopy. Biomed Opt Express, 2013. 4(11): p. 2477-2490.], Ultramarine [Pettikiriarachchi A, G. L., Perugini MA, Devenish RJ, Prescott M, Ultramarine, a chromoprotein acceptor for resonance energy transfer. Sci Rep, 2015. 5.], ShadowY [Murakoshi H, S. A., Nakahata Y, Nabekura J, ShadowY: a dark yellow fluorescent protein for FLIM-based FRET measurement. Sci Rep, 2017. 7(1).], sREACh [Murakoshi H, L. S., Ryohei Yasuda R, Highly sensitive and quantitative FRET-FLIM imaging in single dendritic spines using improved non-radiative YFP. Brain Cell Biol, 2008. 36(1-4): p. 31-42.], E2-Crimson NF [Laufer J, J. A., Pule M, Beard P, In vitro characterization of genetically expressed absorbing proteins using photoacoustic spectroscopy. Biomed Opt Express, 2013. 4(11): p. 2477-2490.], Ultramarine [Pettikiriarachchi A, G. L., Perugini MA, Devenish RJ, Prescott M, Ultramarine, a chromoprotein acceptor for

[0019] The existing dark fluorescent protein almost covers the light region of 424nm to 702nm, however, there is a blank in the light region of 521nm to 572nm, and currently there is no dark fluorescent protein existing in the spectral region of 521nm-572nm. In theory, the dark fluorescent protein in the spectral region of 521nm-572nm can construct a fluorescence resonance energy transfer probe based on fluorescence lifetime imaging with multiple performance superior fluorescent proteins with emission peaks in the green, yellow and orange regions, and has wide application prospects in the fields of biology, medicine, drug screening and the like. SUMMARY

[0020] In order to solve the problems in the prior art, the purposes of the present application are as follows: (1) to develop a new type of high-performance dark fluorescent protein with high extinction coefficient and extremely low quantum yield, and the excitation peak is in the light region of 521nm to 572nm, filling the blank of dark fluorescent protein in the light region; (2) there are many factors determining the brightness of fluorescent protein, and a large number of unknown principles, and the present application aims to find the key switch amino acids determining the brightness of dark fluorescent protein.

[0021] The specific technical scheme of the present application is as follows:

[0022] In one aspect, the present application provides a dark fluorescent protein darkmRuby, and the amino acid sequence of the dark fluorescent protein darkmRuby is shown as SEQ ID NO: 1.

[0023] In another aspect, the present application provides a nucleotide sequence encoding the dark fluorescent protein darkmRuby.

[0024] In still another aspect, the present application provides a vector comprising the nucleotide sequence encoding the dark fluorescent protein darkmRuby.

[0025] In still another aspect, the present application provides brightness-determining amino acids of the dark fluorescent protein darkmRuby, and the brightness-determining amino acids are methionine at the 94th position and phenylalanine at the 96th position of the dark fluorescent protein darkmRuby, and / or phenylalanine at the 174th position of the dark fluorescent protein darkmRuby.

[0026] Further, when the brightness-determining amino acids are methionine at the 94th position and phenylalanine at the 96th position of the dark fluorescent protein darkmRuby are simultaneously mutated into threonine and proline, and / or phenylalanine at the 174th position of the dark fluorescent protein darkmRuby is mutated into histidine, the brightness of the dark fluorescent protein darkmRuby is increased.

[0027] In still another aspect, the present application provides the use of the dark fluorescent protein darkmRuby in preparing a fluorescence resonance energy transfer acceptor or donor based on fluorescence lifetime imaging.

[0028] Further, when the dark fluorescent protein darkmRuby is used as a fluorescence resonance energy transfer acceptor based on fluorescence lifetime imaging, a fluorescent protein with an emission peak in the green, yellow or orange light region is used as a fluorescence resonance energy transfer donor based on fluorescence lifetime imaging.

[0029] In still another aspect of the present application, a protein pair for preparing a fluorescence resonance energy transfer probe based on fluorescence lifetime imaging is provided, comprising the dark fluorescent protein darkmRuby and a fluorescent protein with an emission peak in the green, yellow or orange light region.

[0030] The present application has the following beneficial effects:

[0031] The present application screens a novel high-performance dark fluorescent protein darkmRuby through fluorescent protein engineering and mutation, which has similar spectral properties and similar high extinction coefficient to the starting protein mRuby3, and its quantum yield is reduced to the dark fluorescent protein range (0.05). The excitation peak of darkmRuby is 556 nm, which is the first dark fluorescent protein in the 550-570 light region (orange-red light region).

[0032] The present application finds that the key switch amino acids determining the brightness of darkmRuby are the 94th methionine (Met, M) and the 96th phenylalanine (Phe, F) of darkmRuby, and when the two amino acids are simultaneously mutated into threonine (Thr, T) and proline (Tyr, Y), the dark fluorescent protein property can be removed.

[0033] The present application also finds that when the 174th phenylalanine (Phe, F) of darkmRuby is mutated into histidine (His, H), the brightness of darkmRuby can be significantly increased. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 (a) mRuby3 crystal structure (PDB number: 3U0M) and mutation site diagram of darkmRuby; (b) excitation and emission spectra of darkmRuby and natural light photo; (c) comparison of pH stability of mRuby3 and darkmRuby.

[0035] Figure 2 Comparison diagram of amino acid sequences of mRuby, mRuby2, mRuby3 and darkmRuby.

[0036] Figure 3 HPLC result diagram of mRuby3 and darkmRuby at a concentration of 100 μM. The later the peak appears (i.e. the larger the volume when the peak appears), the stronger the monomer is.

[0037] Figure 4 Comparison of brightness, color, and pH stability between darkmRuby and darkmRuby-TY. (a) Photos of darkmRuby and darkmRuby-TY expressed in E. coli under fluorescence (left) and natural light (right); (b) Comparison of pH stability between darkmRuby, darkmRuby-TY, and darkmRuby-HS.

[0038] Figure 5 Comparison of brightness and color between darkmRuby and darkmRuby-F174H. The images show darkmRuby and darkmRuby-F174H expressed in E. coli under fluorescence (left) and natural light (right). DETAILED DESCRIPTION

[0039] To more clearly understand the present invention, the present invention is further described with reference to the following examples and accompanying drawings. The examples are intended to illustrate the present invention only and are not intended to limit the present invention in any way. In the examples, all raw materials and reagents are commercially available. Experimental methods without specific conditions are conventional methods and conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.

[0040] Example 1 Structure and photophysical properties of the new orange-red dark fluorescent protein darkmRuby

[0041] (1) Crystal structure analysis of orange-red fluorescent protein mRuby3 (Appendix Figure 1 a) and compared with homologous sequences (Appendix Figure 2 ), rationally designed and site-directed mutagenesis were performed on key sites affecting the quantum yield of fluorescent proteins and their interacting amino acids. Mutants were then expressed and screened in the constitutive expression vector pNCS using the Stellar strain. To ensure library integrity, 10 clones were selected for each mutant. Finally, the fluorescence properties of the mutants were determined visually and through blue LED excitation light through an orange acrylic filter. Single clones expressing fluorescent proteins with significantly reduced brightness were screened. Mutants with reduced fluorescence brightness but a high extinction coefficient were then used as templates for the next round of library screening. After multiple rounds of screening, a single clone with extremely low quantum yield was obtained and named darkmRuby. It was based on mRuby3 and had 21 mutations at the sites (N8E, R10P, M15L, S18T, I70V, Y72H, A74K, D75G, F79Y, T94M, Y96F, E111Q, E114V, V116I, V119A, M147I, I160M, N173S, T177E, H193Y, V195I) (Appendix Figure 1 a and attached Figure 2 ).

[0042] The amino acid sequence of darkmRuby (SEQ ID NO: 1) is as follows:

[0043] MVSKGEELIKEEMPMKVVLEGSVNGHQFKCTGEGEGRPYEGVQTMRIKVIEGGPLPFAFDILATSFMYGSRTFVKHPKGIPDYFKQSFPEGFTWERVMRFEDGGVVTVTQDTSLQDGV LIYNAKVRGVNFPSNGPVMQKKTKGWEPNTEMIYPADGGLRGYTDMALKVDGGGHLHCSFVTEYRSKKTVGNIKMPGVYAIDHRLERIEESDNETYVVQREVAVAKYSNLGGGMDELYK

[0044] (2) Bacteria expressing darkmRuby were lysed using B-PER II (purchased from Pierce), and then the protein was purified using HisPur Cobalt Resin (purchased from Pierce), followed by desalting using an Econo-Pac 10DG gravity flow chromatography column (purchased from Bio-Rad, USA). After completing the above protein purification steps, the single-photon excitation spectrum and emission spectrum of darkmRuby were detected using Lambda35 UV / VIS and LS-55 fluorescence spectrometers (purchased from Perkin Elmer). As shown in Table 1 and Appendix Figure 1 As shown in b, the excitation peak of darkmRuby is 556 nm and the emission peak is 597 nm, which is similar to that of mRuby3 (see Appendix Figure 1 b) Its extinction coefficient at the peak is 95mM -1 cm -1 , the quantum yield is 0.05 (Table 1), and the molecular brightness is 4.9, which is determined to be a dim fluorescent protein with high extinction coefficient and low quantum yield.

[0045] (3) The purified darkmRuby protein was concentrated to a high concentration of 5 mg / ml and the fluorescence readings emitted by it in buffers with different pH values ​​when excited by the same wavelength of excitation light were detected using Lambda35 UV / VIS and LS-55 fluorescence spectrometers (purchased from Perkin Elmer). The pKa value of the protein was calculated based on the fluorescence readings. Figure 1 As shown in Figure c, darkmRuby has pKa values ​​of 5.6 and 7.6, showing dual pKa pH-sensitive properties. Its fluorescence brightness reaches its highest at pH 6.5.

[0046] (4) The purified darkmRuby protein was concentrated to a high concentration of 100 mM, and a high-performance liquid chromatograph (Shimadzu LC20A) was used for chromatographic analysis to detect the monomer of the protein. As shown in FIG. 11a, darkmRuby is a monomer at a high concentration, and its monomer is better than that of the known monomer red fluorescent protein FusionRed and mRuby3. Figure 3

[0047] Example 2 Brightness recovery mutant of dark fluorescent protein darkmRuby

[0048] (1) When the methionine (Met, M) at position 94 and the phenylalanine (Phe, F) at position 96 of darkmRuby are simultaneously mutated to threonine (Thr, T) and proline (Tyr, Y) (i.e., M94T&F96Y mutant), the fluorescence brightness and pH alkaline sensitivity are both recovered to a certain extent. This indicates that the amino acids that have a key impact on the properties of darkmRuby dark fluorescent protein are: methionine (Met, M) at position 94 and phenylalanine (Phe, F) at position 96.

[0049] (2) The M94T&F96Y mutant is named darkmRuby-TY, and its single-photon excitation peak is 558 nm and the emission peak is 597 nm (Table 1). Its extinction coefficient at the peak is 64 mM -1 cm -1 , the quantum yield is 0.4, and the molecular brightness is 25.8 (FIG. 11a, Table 1), which has already deviated from the category of dark fluorescent proteins. Figure 4

[0050] (3) The pKa values of darkmRuby-TY are 5.3, 10.4, and 10.8 (FIG. 11b, Table 1), which presents a 3pka characteristic. Compared with darkmRuby, it is more resistant to alkaline environment, and the fluorescence brightness reaches the highest at pH 7. Figure 4

[0051] (4) Mutating the phenylalanine (Phe, F) at position 174 to histidine (His, H) can also significantly increase the brightness of darkmRuby (FIG. 11c). Figure 5

[0052] Table 1 Performance characterization of related fluorescent proteins

[0053]

[0054] Note: a Brightness is calculated by the product of peak EC and QY

[0055] ​​​​Obviously, the above embodiments are merely example for clearly illustrating but not limitation to the embodiments. Based on the above description, other different forms of changes or variations can be made by those skilled in the art. Here, all the embodiments need not and can not be enumerated. The obvious changes or variations derived from the above description are still within the protection scope of the present application.

Claims

1. A dark fluorescent protein darkmRuby, characterized in that, The amino acid sequence of the darkmRuby shown is shown as SEQ ID NO:

1.

2. A nucleotide sequence encoding the darkmRuby of claim 1.

3. A vector, characterized in that, comprising the nucleotide sequence of claim 2.

4. Use of the darkmRuby of claim 1 in the preparation of a fluorescence resonance energy transfer acceptor or donor based on fluorescence lifetime imaging.

5. Use according to claim 4, characterized in that, When the darkmRuby is used as a fluorescence resonance energy transfer acceptor based on fluorescence lifetime imaging, a fluorescent protein with an emission peak in the green, yellow or orange light region is used as a fluorescence resonance energy transfer donor based on fluorescence lifetime imaging.

6. A protein pair for preparing a fluorescence resonance energy transfer probe based on fluorescence lifetime imaging, characterized in that, comprising the darkmRuby of claim 1 and a fluorescent protein with an emission peak in the green, yellow or orange light region.

Images