Ultra-stable red fluorescent protein

Abstract

Disclosed in the present invention is an ultra-stable red fluorescent protein. The fluorescent protein disclosed in the present invention is a protein having an amino acid sequence of SEQ ID NO: 4. Experiments have demonstrated that the fluorescent protein has great tolerance to electron microscopy sample preparation, can retain more fluorescence signals after an osmium tetroxide treatment and can tolerate embedding in an Epon resin. Moreover, the fluorescent protein has great thermal stability and can retain more fluorescence signals even at 90°C. In addition, the fluorescent protein has relatively high chemical stability and light stability. On the basis of related properties of the fluorescent protein, the protein can be used in the fields of protein labeling, various types of fluorescence imaging, and super-resolution correlative light and electron microscopy, and used for tracking changes in structures and morphologies of samples such as proteins, subcellular organelles, local regions of a cell, and entire cells. The fluorescent protein can be used for packaging related viruses or generate transgenic animals, and for labeling specific proteins, specific organelles or specific cells, thereby achieving rapid tissue clearing imaging and expansion super-resolution microscopy.

Description

An ultra-stable red fluorescent protein Technical Field

[0001] This invention relates to an ultrastable red fluorescent protein in the field of bioimaging. Background Technology

[0002] Fluorescent proteins enable the visualization, tracking, and quantification of molecules and events in living cells with high spatial and temporal resolution, thus revolutionizing the applications of microscopic imaging. Among all these properties, the stability of fluorescent proteins plays a crucial role in microscopic imaging. Recently, the development of StayGold, the most photostable green fluorescent protein to date, and its monomeric variants, has revolutionized long-term live-cell imaging techniques. However, red fluorescent proteins still outperform green fluorescent proteins in terms of autofluorescence, light scattering, and phototoxicity. Therefore, the development of stable red fluorescent proteins is urgently needed.

[0003] Super-resolution photoelectric correlation microscopy (SEM) not only precisely locates target proteins but also provides information about their ultramicroscopic environment. The inventors of mEosEM, a fluorescent protein resistant to conventional electron microscopy sample preparation, developed in 2020, achieved super-resolution SEM after embedding in a high-temperature polymerized hydrophobic resin (Epon). Compared to other super-resolution SEM techniques, Epon-embedded super-resolution SEM better preserves the ultrastructure of the sample, making it more suitable for continuous ultrathin sections and three-dimensional electron microscopy reconstruction. Following mEosEM, scientists have discovered that some existing fluorescent proteins, such as mKate2, mWasabi, CoGFPv0, mCherry2, mEosEM-E, the mScarlet series, and hfYFP, retain fluorescence signals during conventional electron microscopy sample preparation. However, after fixation with 1% osmium tetroxide, the remaining fluorescence signal ratio is only about 10%. If embedded in Epon, the retained fluorescence signal will be even lower. Therefore, more fluorescent proteins resistant to conventional electron microscopy sample preparation are needed to broaden the biological applications of super-resolution SEM.

[0004] Tissue clearing technology is a key element in the realization of modern three-dimensional fluorescence imaging of biological tissues because it makes biological tissues transparent, enabling visualization of deep and complex tissue structures. These methods involve removing lipids and other light-scattering components from the tissue, allowing light to penetrate deeper and reducing light scattering. Due to limitations in the thermal stability of fluorescent proteins, tissue clearing is performed at room temperature or below 37°C to minimize the loss of fluorescence signal caused by drastic temperature changes. Therefore, the tissue clearing process is extremely time-consuming, typically requiring one to several weeks to achieve optimal transparency. Increasing the temperature could potentially accelerate the tissue clearing process, but this might affect the stability of the fluorescent proteins. Previously, green thermostable fluorescent proteins have been successfully developed. Despite these advances, there remains a significant need for red thermostable fluorescent proteins to accelerate the tissue clearing process without compromising image quality.

[0005] Structured illumination imaging (SIM) is widely considered the preferred super-resolution imaging method for live-cell imaging due to its high imaging efficiency and low phototoxicity. Compared with two-dimensional structured illumination imaging, three-dimensional structured illumination imaging places more stringent requirements on the photostability of fluorescent probes due to its unique imaging process. First, three-dimensional structured illumination imaging captures and merges 15 images per frame (5 phases × 3 angles), significantly more than two-dimensional structured illumination imaging. Second, during three-dimensional imaging, fluorescent probes above and below the focal plane are simultaneously illuminated. This omnidirectional excitation further exacerbates the risk of photobleaching of fluorescent probes. Although StayGold and its variants have been reported to achieve long-duration single-channel three-dimensional structured illumination imaging, there is a lack of a red photostable fluorescent protein that can be co-labeled with StayGold to achieve long-duration three-dimensional structured illumination imaging.

[0006] In stimulated emission depletion super-resolution imaging (STED), a tightly focused excitation beam scans the sample using a grating, followed by a donut-shaped depletion beam. This imaging method effectively reduces the excitation spot size. However, it places extremely high demands on the photostability of the fluorescent probes. While organic dyes are commonly used in STED due to their photostability, fluorescent probes offer significant advantages in specificity and gene targeting. Therefore, there is currently a significant need for red photostable fluorescent proteins to expand the spectral range of fluorescent probes used in STED microscopy. Summary of the Invention

[0007] The core technical problem to be solved by this invention is how to improve the photothermal stability, osmium acid resistance and chemical stability of red fluorescent protein.

[0008] To address the aforementioned technical problems, this invention first provides a non-naturally derived protein (synthetic construct) named mBaoHong, which is as follows: A1), A2), or A3):

[0009] A1) A protein whose amino acid sequence includes SEQ ID NO:4;

[0010] A2) A protein that has the same function as the amino acid sequence shown in SEQ ID NO:4 in the sequence listing, but with one or more amino acid residues replaced and / or deleted and / or added, while keeping the amino acid residue at position 163 unchanged.

[0011] A3) is a fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of A1) or A2).

[0012] The protein mentioned in A1) above may specifically be a protein with the amino acid sequence SEQ ID NO:4.

[0013] The protein in A2) above is a protein that has 75% or more amino acid sequence identity with the protein shown in SEQ ID NO:4 and has the same function. The 75% or more identity can be at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity.

[0014] The determination of amino acid sequence identity percentages can be achieved through various known methods, such as using publicly available computer software like BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR). Appropriate parameters for sequence alignment can be determined, including the algorithms required to achieve maximum alignment across the entire length of the sequences being compared. However, for the purposes of this paper, the sequence comparison computer program ALIGN-2 is used to generate amino acid sequence identity percentage values. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and its source code has been submitted with user documentation to the U.S. Copyright Office, Washington, D.C., 20559, under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc. (South San Francisco, California) or can be compiled from the source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not change.

[0015] The present invention also provides mBaoHong-related biomaterials, said biomaterials being any one of the following B1) to B9):

[0016] B1) Nucleic acid molecules encoding mBaoHong;

[0017] B2) An expression cassette containing the nucleic acid molecule described in B1);

[0018] B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0019] B4) A viral vector containing the nucleic acid molecule described in B1), or a viral vector containing the expression cassette described in B2), or a viral vector containing the recombinant vector described in B3);

[0020] B5) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3);

[0021] B6) A transgenic cell line containing the nucleic acid molecule described in B1), or a transgenic cell line containing the expression cassette described in B2);

[0022] B7) Transgenic tissue containing the nucleic acid molecules described in B1), or transgenic tissue containing the expression cassette described in B2);

[0023] B8) A transgenic organ containing the nucleic acid molecule described in B1), or a transgenic organ containing the expression cassette described in B2);

[0024] B9) Transgenic animals containing the nucleic acid molecules described in B1) or transgenic animals containing the expression cassette described in B2).

[0025] In the above-mentioned biological materials, the nucleic acid molecule described in B1) may be as follows: b11), b12), or b13):

[0026] b11) The coding sequence is the cDNA molecule or DNA molecule of SEQ ID NO:3 in the sequence listing;

[0027] b12) The cDNA molecule or DNA molecule shown in SEQ ID NO:3 in the sequence listing;

[0028] b13) has 75% or more identity with the nucleotide sequence defined by b11) or b12) and encodes a cDNA molecule or DNA molecule of mBaoHong.

[0029] The nucleic acid molecule is of non-natural origin and is a synthetic construct. The nucleic acid molecule can be DNA, such as cDNA, genomic DNA, or recombinant DNA; it can also be RNA, such as mRNA or hnRNA.

[0030] Those skilled in the art can readily mutate the nucleotide sequence encoding the protein of the present invention using known methods, such as directed evolution and point mutation. Artificially modified nucleotides that have 75% or higher identity with the nucleotide sequence of the protein isolated from the present invention, as long as they encode the protein shown in SEQ ID NO:4, retain the same amino acid residue at position 163 as SEQ ID NO:4, and have the same protein function, are all derived from and equivalent to the nucleotide sequence of the present invention.

[0031] As used herein, the term "identity" refers to sequence similarity to a natural nucleic acid sequence. "Identity" includes nucleotide sequences having 75% or higher, 85% or higher, 90% or higher, or 95% or higher identity with the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:4 of this invention. Identity can be evaluated visually or using computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0032] The expression cassette containing a nucleic acid molecule encoding mBaoHong, as described in B2), refers to DNA capable of expressing mBaoHong in host cells. This DNA may include not only a promoter to initiate transcription of the mBaoHong-encoding gene but also a terminator to terminate transcription of the mBaoHong-encoding gene. Furthermore, the expression cassette may also include an enhancer sequence.

[0033] Recombinant vectors containing the mBaoHong gene expression cassette can be constructed using existing expression vectors.

[0034] The vector may be a plasmid, granule, bacteriophage, or viral vector. Specifically, the plasmid may be the pET-28a(+) vector.

[0035] B3) The recombinant vector may specifically be pET-28a(+)-mBaoHong, where pET-28a(+)-mBaoHong is a recombinant vector obtained by replacing the DNA fragment between the BamHI and NotI recognition sequences of the pET-28a(+) vector with the mBaoHong gene.

[0036] The microorganism may be yeast, bacteria, algae, or fungi. The bacteria may be Escherichia coli.

[0037] The cells can be plant cells or animal cells. In one embodiment of the invention, HeLa cells are used as a reference example.

[0038] The transgenic cell lines, transgenic tissues, and transgenic organs may be animal transgenic cell lines, transgenic tissues, and transgenic organs, or they may be plant transgenic cell lines, transgenic tissues, and transgenic organs. The transgenic cell lines, transgenic tissues, transgenic organs, and transgenic animals may or may not include reproductive material.

[0039] The application of mBaoHong as a fluorescent protein is also within the scope of protection of this invention.

[0040] The present invention also provides a method for locating a target protein, the method comprising: linking the coding gene of mBaoHong with the coding gene of the target protein and introducing it into an isolated target cell, target tissue, target organ or target individual, thereby causing the target cell, the target tissue, the target organ or the target individual to express a fusion protein formed by mBaoHong and the target protein, detecting the fluorescence signal of mBaoHong in the target cell, the target tissue, the target organ or the target individual, thereby achieving the localization of the target protein.

[0041] In the above method, the encoding gene of mBaoHong and the encoding gene of the target protein can be introduced into the target cell, the target tissue, the target organ, or the target individual through an expression vector containing the encoding gene of mBaoHong and the encoding gene of the target protein.

[0042] In the above method, the target cells, target tissues, target organs, or target individuals can be embedded using the Epon resin embedding method.

[0043] The method can employ osmium tetroxide fixation to fix the target cells, target tissues, target organs, or target individuals.

[0044] In the above method, the environment in which the target cell, target tissue, target organ, or target individual is located can be 90°C or below. Further, the environment can be 60°C-90°C or below. Further, the environment can be below 89°C, below 88°C, below 87°C, below 86°C, below 85°C, below 84°C, below 83°C, below 82°C, below 81°C, below 80°C, below 79°C, below 78°C, below 77°C, below 76°C, below 75°C, below 74°C, below 73°C, below 72°C, below 71°C, below 70°C, below 69°C, below 68°C, below 67°C, below 66°C, below 65°C, below 64°C, below 63°C, below 62°C, below 61°C, 60°C, 89°C-90°C, 88°C-89°C, 87°C-88°C, 86°C, or below 89°C. ℃-87℃, 85℃-86℃, 84℃-85℃, 83℃-84℃, 82℃-83℃, 81℃-82℃, 80℃-81℃, 79℃-80℃, 78℃-79℃, 77℃-78℃, 76℃-77℃, 75℃-76℃, 74℃-75℃, 73℃-74℃, 72℃-73℃, 71℃-72℃, 70℃-71℃, 69℃-70℃, 68℃-69℃, 67℃-68℃, 66℃-67℃, 65℃-66℃, 64℃-65℃, 63℃-64℃, 62℃-63℃, 61℃-62℃ or 60℃-61℃.

[0045] The present invention also provides a method for locating a target protein, the method comprising: 1) providing a gene encoding a fusion protein, the fusion protein comprising the mBaoHong and the target protein;

[0046] 2) The gene encoding the fusion protein is introduced into the target cell, target tissue, target organ, or target individual, so that the target cell, target tissue, target organ, or target individual expresses the fusion protein, and the fluorescence signal of mBaoHong in the target cell, target tissue, target organ, or target individual is detected to achieve the localization of the target protein.

[0047] Locating the target protein means determining the location where the target protein is present.

[0048] Chromogenic agents containing mBaoHong or the aforementioned biological materials are also within the scope of protection of this invention.

[0049] The present invention also provides any of the following applications of mBaoHong or the biomaterial:

[0050] X1) Protein markers;

[0051] X2) fluorescence imaging;

[0052] X3) Photoelectric correlation microscopy;

[0053] X4) Track the structure and / or morphology of proteins, subcellular organelles, localized cellular regions, cells, or small animal embryos;

[0054] X5) Analyze the structure and / or location of the target protein;

[0055] X6) Prepare protein-labeled products;

[0056] X7) Prepare fluorescence imaging products;

[0057] X8) Prepare photoelectric correlation microscopic imaging products;

[0058] X9) Prepare products that track the structure and / or morphology of proteins, subcellular organelles, localized cellular regions, cells, or small animal embryos;

[0059] X10) Prepare products for analyzing the structure and / or localization of the target protein;

[0060] X11) Prepare relevant viruses or transgenic animals, label specific proteins, specific cells or specific organelles to achieve rapid transparent tissue imaging and expanded super-resolution imaging.

[0061] The aforementioned fluorescence imaging includes conventional fluorescence imaging and ultra-high resolution fluorescence imaging.

[0062] The aforementioned traditional fluorescence imaging includes, but is not limited to, wide-field fluorescence imaging, confocal microscopy, total internal reflection fluorescence imaging, and live-cell fluorescence imaging.

[0063] The aforementioned ultra-high resolution fluorescence imaging includes, but is not limited to, optical wave ultra-high resolution imaging (SOFI), structured light illumination imaging (SIM), nonlinear structured light illumination imaging (PANL-SIM), stimulated emission loss ultra-high resolution imaging (STED), and reversible saturable optical fluorescence transition ultra-high resolution imaging (RESOLFT).

[0064] The aforementioned photoelectric correlation microscopy can be classified as super-resolution photoelectric correlation microscopy.

[0065] The fluorescence imaging in the aforementioned photoelectric correlation microscopy includes, but is not limited to, wide-field fluorescence imaging, confocal microscopy, total internal reflection fluorescence imaging, live-cell fluorescence imaging, optical wave super-resolution imaging (SOFI), structured light illumination imaging (SIM), nonlinear structured light illumination imaging (PANL-SIM), stimulated emission loss super-resolution imaging (STED), and reversible saturable optical fluorescence transition super-resolution imaging (RESOLFT).

[0066] The electron microscopy imaging in the aforementioned photoelectric correlation microscopy includes, but is not limited to, scanning electron microscopy, transmission electron microscopy, and cryo-electron microscopy. The sample embedding methods for electron microscopy include, but are not limited to, embedding methods using hydrophobic resins, hydrophilic resins, and low-temperature resins.

[0067] The application of the fluorescent protein of the present invention in photoelectric correlation microscopy includes, but is not limited to, two-dimensional light-electron microscopy combined imaging, three-dimensional light-electron microscopy combined imaging, and three-dimensional continuous slice light-electron microscopy combined imaging.

[0068] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way. Attached Figure Description

[0069] Figure 1 shows the photostability test of the fluorescent protein. **** indicates that the significance analysis reached p < 0.00001.

[0070] Figure 2 shows the thermal stability of the fluorescent protein. The protein was incubated at 5°C intervals for 1 hour within the temperature range of 60°C to 90°C.

[0071] Figure 3 shows the osmium tetroxide resistance test of the fluorescent protein. The osmium tetroxide resistance test time was 10 min. **** indicates that the significance analysis reached p < 0.00001.

[0072] Figure 4 shows the percentage of fluorescence retention under a fluorescence microscope after electron microscopy preparation of HeLa cells transfected with fluorescent proteins. mScarlet-3 is mScarlet3. **** indicates that the significance analysis reached p < 0.00001.

[0073] Figure 5 shows the chemical stability test of the fluorescent protein. Here, pH represents the change in pH value of the system after adding the corresponding concentrations of guanidine hydrochloride, guanidine thiocyanate, or urea. Embodiments of the present invention

[0074] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials, reagents, and instruments used in the following examples are commercially available. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged. Unless otherwise specified, in the following examples, the first position of each nucleotide sequence in the sequence listing is the 5′ terminal nucleotide of the corresponding DNA, and the last position is the 3′ terminal nucleotide of the corresponding DNA.

[0075] Preparation of the mito-mCherry2 vector: The DNA fragment between the NheI and AgeI recognition sequences of the pEGFP-N1 vector was replaced with the mitochondrial localization sequence to obtain the recombinant vector mito-EGFP; the DNA fragment containing the EGFP gene between the AgeI and NotI recognition sequences of mito-EGFP was replaced with the mCherry2 gene to obtain the recombinant vector mito-mCherry2.

[0076] The mitochondrial localization sequence is as follows:

[0077] The mCherry2 gene sequence is as follows:

[0078] Example 1: mBaoHong is a fluorescent protein with good photothermal stability and strong fluorescence signal.

[0079] I. Generation of mutants and construction of recombinant vectors

[0080] Based on the publicly available protein sequence information of the fluorescent protein mScarlet3, the gene sequence of mScarlet3 was artificially synthesized and constructed into a mitochondrial-targeting vector to obtain mito-mScarlet3. The methionine residue at position 163 of mScarlet3 was mutated to a histidine residue to obtain mBaoHong, and the recombinant vector containing the mBaoHong gene is denoted as mito-mBaoHong.

[0081] The amino acid sequences of mScarlet3 and mBaoHong are shown in SEQ ID NO:2 and SEQ ID NO:4 in the sequence listing, respectively, and the DNA sequences of the mScarlet3 gene and mBaoHong gene are shown in SEQ ID NO:1 and SEQ ID NO:3 in the sequence listing, respectively.

[0082] Construction of recombinant vectors:

[0083] mito-mScarlet3: The recombinant vector mito-mScarlet3 is obtained by replacing the DNA fragment between the AgeI and NotI recognition sequences of the mito-mCherry2 vector with the mScarlet3 gene. This recombinant vector can express the mScarlet3 protein.

[0084] mito-mBaoHong: The recombinant vector obtained by replacing the DNA fragment between the AgeI and NotI recognition sequences of the mito-mCherry2 vector with the mBaoHong gene is mito-mBaoHong, which can express the mBaoHong protein.

[0085] mito-mScarlet-H: The recombinant vector mito-mScarlet-H is obtained by replacing the DNA fragment between the AgeI and NotI recognition sequences of the mito-mCherry2 vector with the mScarlet-H gene. This recombinant vector can express the mScarlet-H protein.

[0086] mScarlet-H gene:

[0087] mScarlet-H protein:

[0088] mito-mScarlet-I3: The recombinant vector mito-mScarlet-I3 is obtained by replacing the DNA fragment between the AgeI and NotI recognition sequences of the mito-mCherry2 vector with the mScarlet-I3 gene. This recombinant vector can express the mScarlet-I3 protein.

[0089] mScarlet-I3 gene:

[0090] mScarlet-I3 protein:

[0091] mito-mScarlet: The recombinant vector obtained by replacing the DNA fragment between the AgeI and NotI recognition sequences of the mito-mCherry2 vector with the mScarlet gene is mito-mScarlet, which can express the mScarlet protein.

[0092] mScarlet gene:

[0093] mScarlet protein:

[0094] mito-mScarlet-I: The recombinant vector mito-mScarlet-I is obtained by replacing the DNA fragment between the AgeI and NotI recognition sequences of the mito-mCherry2 vector with the mScarlet-I gene. This recombinant vector can express the mScarlet-I protein.

[0095] mScarlet-I gene:

[0096] mScarlet-I protein:

[0097] mito-oScarlet: The recombinant vector obtained by replacing the DNA fragment between the AgeI and NotI recognition sequences of the mito-mCherry2 vector with the oScarlet gene is mito-oScarlet, which can express the oScarlet protein.

[0098] oScarlet gene:

[0099] oScarlet protein:

[0100] II. Determination of Fluorescent Protein Properties

[0101] 1. Construction of recombinant vectors

[0102] The mScarlet, mScarlet-I, oScarlet, mScarlet-H, mScarlet-I3, mScarlet3, and mBaoHong genes were constructed into the pET-28a(+) vector, as detailed below:

[0103] The DNA fragment between the BamHI and NotI recognition sequences of the pET-28a(+) vector was replaced with the mScarlet3 gene, and the resulting recombinant vector was designated pET-28a(+)-mScarlet3. This recombinant vector contains the mScarlet3 gene shown in SEQ ID NO:1 and can express a fusion protein formed by fusing the C-terminus of a polypeptide containing a 6×His tag (the sequence of which is SEQ ID NO:5 in the sequence listing) with the mScarlet3 protein shown in SEQ ID NO:2.

[0104] The DNA fragment between the BamHI and NotI recognition sequences of the pET-28a(+) vector was replaced with the mBaoHong gene, and the resulting recombinant vector was designated pET-28a(+)-mBaoHong. This recombinant vector contains the mBaoHong gene shown in SEQ ID NO:3 and can express a fusion protein formed by fusing the C-terminus of a polypeptide containing a 6×His tag (the sequence of which is SEQ ID NO:5 in the sequence listing) with the mBaoHong protein shown in SEQ ID NO:4.

[0105] The DNA fragment between the BamHI and NotI recognition sequences of the pET-28a(+) vector was replaced with the mScarlet-H gene, and the resulting recombinant vector was designated as pET-28a(+)-mScarlet-H. This recombinant vector contains the mScarlet-H gene and can express a fusion protein formed by fusing the C-terminus of a polypeptide containing a 6×His tag (the sequence of which is SEQ ID NO:5 in the sequence listing) with the mScarlet-H protein.

[0106] The DNA fragment between the BamHI and NotI recognition sequences of the pET-28a(+) vector was replaced with the mScarlet-I3 gene, and the resulting recombinant vector was designated pET-28a(+)-mScarlet-I3. This recombinant vector contains the mScarlet-I3 gene and can express a fusion protein formed by fusing the C-terminus of a polypeptide containing a 6×His tag (the sequence of which is SEQ ID NO:5 in the sequence listing) with the mScarlet-I3 protein.

[0107] The DNA fragment between the BamHI and NotI recognition sequences of the pET-28a(+) vector was replaced with the mScarlet gene, and the resulting recombinant vector was named pET-28a(+)-mScarlet. This recombinant vector contains the mScarlet gene and can express a fusion protein formed by fusing the C-terminus of a polypeptide containing a 6×His tag (the sequence of which is SEQ ID NO:5 in the sequence listing) with the mScarlet protein.

[0108] The DNA fragment between the BamHI and NotI recognition sequences of the pET-28a(+) vector was replaced with the mScarlet-I gene, and the resulting recombinant vector was designated as pET-28a(+)-mScarlet-I. This recombinant vector contains the mScarlet-I gene and can express a fusion protein formed by fusing the C-terminus of a polypeptide containing a 6×His tag (the sequence of which is SEQ ID NO:5 in the sequence listing) with the mScarlet-I protein.

[0109] The DNA fragment between the BamHI and NotI recognition sequences of the pET-28a(+) vector was replaced with the oScarlet gene, and the resulting recombinant vector was designated pET-28a(+)-oScarlet. This recombinant vector contains the oScarlet gene and can express a fusion protein formed by fusing the C-terminus of a polypeptide containing a 6×His tag (the sequence of which is SEQ ID NO:5 in the sequence listing) with the oScarlet protein.

[0110] 2. Expression and purification of fluorescent proteins

[0111] Expression of fluorescent proteins:

[0112] The recombinant vectors from step 1 were introduced into BL21 *E. coli* to obtain the corresponding recombinant bacteria. The recombinant bacteria were then used to express fluorescent proteins according to the following steps: Recombinant bacteria were inoculated into 1 ml of LB medium containing kanamycin at a standard concentration of 1 ng / 100 μL. After the bacterial culture became turbid, it was expanded to 100 mL of LB medium containing kanamycin and cultured at 37°C with shaking until the OD value of the bacterial culture reached approximately 0.6-0.8. The culture was then complete. IPTG was added to a final concentration of 1 mM, and the culture was continued at 16°C for 16-20 h to induce a large amount of protein expression.

[0113] Purification of fluorescent proteins:

[0114] After protein expression induction, the resulting bacterial culture was centrifuged at 7000 rpm for 5 min at 4°C, the supernatant was discarded, and the bacterial cells were collected. After collection, the cells were washed and resuspended once with binding buffer (20 mM Tris-HCl pH = 7.4, 150 mM NaCl) and centrifuged again. The bacterial cells were resuspended again with 10 mL of binding buffer and subjected to sonication in an ice-water bath (2 s sonication, 4 s pause, total sonication time 30 min; power: 60%). After sonication, the cells were centrifuged at 10000 rpm for 30 min at 4°C, and the protein supernatant was collected.

[0115] To prepare the nickel column, first elute the ethanol from the column, wash with ddH2O for 25 column volumes, add 5 column volumes of 0.2M NiSO4·6H2O, wash again with ddH2O for 25 column volumes, and finally wash with binding buffer for 5 column volumes. Transfer the protein supernatant to the pre-treated nickel column and incubate in a silent mixer at 4°C for 2-3 hours. After incubation, perform a gradient elution to remove contaminating proteins. First, wash with pre-cooled low-concentration imidazole solution (20 mM imidazole, 20 mM Tris-HCl pH=7.4, 150 mM NaCl), then continue washing with 35 mM imidazole solution (35 mM imidazole, 20 mM Tris-HCl pH=7.4, 150 mM NaCl), and finally wash with 40 mM imidazole solution (40 mM imidazole, 20 mM Tris-HCl pH=7.4, 150 mM NaCl). After washing, the target protein was eluted with a 300 mM high-concentration imidazole solution (300 mM imidazole, 20 mM Tris-HCl pH 7.4, 150 mM NaCl) and collected. The protein was then concentrated to approximately 1 mL by centrifugation at 4000 rpm for 10 min at 4°C using a 10 kDa ultrafiltration tube. Imidazole-free binding buffer was then added, and the process was repeated three times under the same conditions. After concentration, the protein concentration was determined using Nanodrop, and 100 μL of the protein was aliquoted into centrifuge tubes and stored at -80°C.

[0116] The protein purification system used was AKTA Pure. First, prepare 1L of ddH2O, binding buffer (20mM Tris-HCl pH=7.4, 150mM NaCl), 0.5M NaOH aqueous solution, and 500mL of 20% ethanol aqueous solution after sonication. Clean the A pump used with AKTA Pure with ddH2O, setting the flow rate to 1mL / min. During flow, connect the Superdex-75Increase 10 / 300GL gel column to the system. Wash the column with ddH2O for one column volume, followed by one column volume with binding buffer at a flow rate of 0.8mL / min and a pressure limit of 5.0MPa. Clean the sample loading tip, load 2mL, and run the pre-set program. Collect the purified protein, clearly label it, concentrate it to 5mg / mL, and store at -80℃. After the procedure, the column was washed sequentially with ddH2O, 0.5M NaOH aqueous solution, ddH2O, and 20% ethanol aqueous solution at a flow rate of 0.8 mL / min for one column volume. The gel column was then stored in 20% ethanol aqueous solution and removed. Finally, pump A was pump washed with 20% ethanol, and the entire system was stored in 20% ethanol aqueous solution before saving the data.

[0117] The purified fusion proteins obtained were mScarlet fusion protein, mScarlet-I fusion protein, oScarlet fusion protein, mScarlet-H fusion protein, mScarlet-I3 fusion protein, mScarlet3 fusion protein, and mBaoHong fusion protein.

[0118] 3. Light stability test

[0119] The recombinant vector mito-mBaoHong was transfected into HeLa cells, and its photostability was compared with that of mScarlet-H, the fluorescent protein with the best photostability and best electron microscopy sample preparation effect, under the same parameter settings. The results are shown in Figure 1. Under the same parameter settings, the photostability of mBaoHong is much higher than that of mScarlet-H (Figure 1a), and the time for the fluorescence to drop to half value is longer than that of mScarlet-H (Figure 1b).

[0120] The parameters are set as follows: select the 561nm excitation channel and set the light intensity to 5.006mW.

[0121] 4. Thermal stability and osmium tetroxide resistance test

[0122] Thermal stability test: The purified fluorescent protein obtained in step 2 was diluted to 0.02 mg / mL with binding buffer (pH = 7.4). The fluorescence signal after incubation at 60℃ to 90℃ for 1 h was detected using a microplate reader. The excitation wavelength was set to 561 nm and the emission wavelength was set to 590 nm. Binding buffer (pH = 7.4) was used as a blank control to eliminate background interference. As shown in Figure 2, the fluorescence retention of mBaoHong after treatment at 90℃ was much higher than that of mScarlet-H and mScarlet3.

[0123] Osmium tetroxide resistance test: Performed at room temperature. The purified fusion protein obtained in step 2 was diluted to 0.02 mg / mL with binding buffer (pH = 7.4), and then OsO4 was added to a final concentration of 1 g / 100 mL. A control was set up (OsO4 was added to binding buffer (pH = 7.4) to a final concentration of 1 g / 100 mL). After 10 minutes, fluorescence was recorded using a microplate reader. The excitation wavelength was set to 561 nm, and the emission wavelength was set to 590 nm. Binding buffer (pH = 7.4) was used as a blank control to eliminate background interference. As shown in Figure 3, the fluorescence retention percentage of mBaoHong after 1% osmium tetroxide treatment was much higher than that of mScarlet-H, mScarlet, mScarlet-I, oScarlet, mScarlet3, and mScarlet-I3.

[0124] 5. Electron microscopy sample preparation and testing

[0125] The recombinant vectors from step one (mito-mScarlet, mito-mScarlet-I, mito-mScarlet3, mito-mScarlet-I3, mito-oScarlet, mito-mScarlet-H, mito-mBaoHong) were transfected into HeLa cells. After 48 hours, the cells were digested and fixed for electron microscopy. Ultrathin sections were then prepared for fluorescence detection and comparison. The specific steps are as follows:

[0126] When HeLa cells are in the logarithmic growth phase and their density reaches approximately 80%, the recombinant vector is transfected into the HeLa cells. To ensure that the fluorescent protein is fully folded and matured, the cells are fixed 48 hours after transfection, and electron microscopy sample preparation begins. The sample preparation steps are as follows:

[0127] a. After tryingpsin digestion, cells that have been transfected with fluorescent protein for 48 hours were centrifuged at 1000×g for 10 min, the culture medium was discarded, and fixative (final concentration 4% PFA + 0.25% GA + 0.01M PBS) was added and incubated overnight at 4℃.

[0128] b. Discard the fixative and wash three times with 0.01M PBS, 10 min each time.

[0129] c. Add 1 g / 100 mL of osmium tetroxide (solvent is Binding buffer (pH=7.4)) and fix at 4°C for 1 h.

[0130] d. Remove the fixative and wash three times with ddH2O for 10 minutes each time.

[0131] e. Add 2% alcohol uranium staining solution (obtained by dissolving uranium in ethanol, wherein the concentration of uranium is 2g / 100ml), and stain with uranium at 4℃ in the dark for 1 hour.

[0132] f. Remove the 2% alcohol uranium stain and wash three times with ddH2O for 10 minutes each time.

[0133] g. Gradient dehydration: Dehydrate using 30%, 50%, 70%, 80%, and 90% ethanol aqueous solutions for 10 minutes each time, followed by two 100% ethanol dehydration cycles for 10 minutes each.

[0134] h, dehydrate with anhydrous acetone 3 times, 10 minutes each time.

[0135] i. Use acetone: treat with permeate solutions of 3:1, 1:1 and 1:3 for 1h, 2h and 3h respectively. Resin formula: Epon 812 11.05g, DDSA 6.1879g, NMA 6.5426g, DMP-30 0.3914g.

[0136] j. Use pure resin to permeate 3 times, 12 hours each time.

[0137] k. Place the resin-coated sample in a 60℃ oven for polymerization for 14-16 hours.

[0138] After polymerization, the resin block was trimmed using an ultrathin slicer, and the sample was cut into ultrathin slices with a thickness of 100 nm. The fluorescence signal of the fluorescent protein sample after electron microscopy was examined using a fluorescence microscope.

[0139] As shown in Figure 4, under the same parameter settings, the 561nm excitation channel was selected and the light intensity was set to 10mW. It can be seen that the fluorescence intensity of mito-mBaoHong sample was the strongest and much higher than that of the template mScarlet3 and other fluorescent proteins, mScarlet, mScarlet-I, oScarlet, mScarlet-3, and mScarlet-I3.

[0140] 6. Chemical stability test

[0141] Chemical stability test: The purified fluorescent protein obtained in step 2 was diluted to 0.02 mg / mL with binding buffer (pH = 7.4). Guanidine hydrochloride, guanidine thiocyanate, or urea was added to the diluted solution. The fluorescence signal of the purified fluorescent protein after treatment with 1M-8M guanidine hydrochloride, 1M-4M guanidine thiocyanate, and 1-10M urea for 16 h was detected using an ELISA reader. The excitation wavelength was set to 561 nm and the emission wavelength was set to 590 nm. Binding buffer (pH = 7.4) was used as a blank control to eliminate background interference. As shown in Figure 5, the fluorescence retention of mBaoHong after treatment at 90℃ was higher than that of mScarlet-H and mScarlet3.

[0142] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Industrial applicability

[0143] Experiments have demonstrated that the protein shown in SEQ ID NO:4 of this invention can serve as a fluorescent protein, exhibiting excellent photothermal stability, resistance to osmium tetroxide, and chemical stability. Based on these characteristics, the fluorescent protein of this invention can be independently used in the fields of protein labeling, fluorescence imaging, live-cell dynamic imaging, and super-resolution photoelectric correlation microscopy. It can be used to track structural and morphological changes in samples such as proteins, subcellular organelles, local cellular regions, cells, and small animal embryos; it can be used to package related viruses or transgenic animals, label specific proteins, specific cells, or specific organelles, and achieve rapid transparent tissue imaging and expanded super-resolution imaging; and it also exhibits high fluorescence intensity after conventional chemical electron microscopy sample preparation. Therefore, the fluorescent protein of this invention has broad application prospects. Sequence List Free Content

[0144]

[0145] Cross-references to related applications

[0146] This application claims priority to Chinese patent application No. 202410568362.4, filed on May 9, 2024, the entire contents of which are incorporated herein by reference.

Claims

1. Protein, in the following forms: A1), A2), or A3): A1) The amino acid sequence is that of the protein SEQ ID NO:4; A2) A protein that has the same function as the amino acid sequence shown in SEQ ID NO:4 in the sequence listing, but with one or more amino acid residues replaced and / or deleted and / or added, while keeping the amino acid residue at position 163 unchanged. A3) is a fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of A1) or A2).

2. The biomaterial relating to the protein of claim 1 is any one of B1) to B8) below: B1) A nucleic acid molecule encoding the protein of claim 1; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) A viral vector containing the nucleic acid molecule described in B1), or a viral vector containing the expression cassette described in B2), or a viral vector containing the recombinant vector described in B3); B5) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3); B6) A transgenic cell line containing the nucleic acid molecule described in B1), or a transgenic cell line containing the expression cassette described in B2); B7) Transgenic tissue containing the nucleic acid molecules described in B1), or transgenic tissue containing the expression cassette described in B2); B8) A transgenic organ containing the nucleic acid molecule described in B1) or a transgenic organ containing the expression cassette described in B2).

3. The biomaterial according to claim 2, characterized in that: B1) The nucleic acid molecule described is as follows: (b11) or (b12) or (b13) b11) The coding sequence is the cDNA molecule or DNA molecule of SEQ ID NO:3 in the sequence listing; b12) The cDNA molecule or DNA molecule shown in SEQ ID NO:3 in the sequence listing; b13) has 75% or more identity with the nucleotide sequence defined by b11) or b12) and encodes a cDNA molecule or DNA molecule of the protein of claim 1.

4. The use of the protein of claim 1 as a fluorescent protein.

5. Methods for locating the target protein, including: The coding gene of the protein described in claim 1 is linked to the coding gene of the target protein and then introduced into the target cell, target tissue, target organ, or target individual, so that the target cell, the target tissue, the target organ, or the target individual expresses the fusion protein formed by the protein described in claim 1 and the target protein, and the fluorescence signal of the protein described in claim 1 in the target cell, the target tissue, the target organ, or the target individual is detected to achieve the localization of the target protein.

6. The method according to claim 5, characterized in that: The protein-coding gene of claim 1 and the target protein-coding gene are introduced into the target cell, the target tissue, the target organ, or the target individual via an expression vector containing the protein-coding gene and the target protein-coding gene.

7. The method according to claim 5 or 6, characterized in that: The method employs Epon resin embedding to embed the target cells, target tissues, target organs, or target individuals.

8. The method according to any one of claims 5-7, characterized in that: The method employs osmium tetroxide fixation to fix the target cells, target tissues, target organs, or target individuals.

9. The method according to any one of claims 5-8, characterized in that: The target cell, the target tissue, the target organ, or the target individual is located in an environment at or below 90°C.

10. A chromogenic agent containing the protein of claim 1 or the biomaterial of claim 2 or 3.

11. Any of the following applications of the protein of claim 1 or the biomaterial of claim 2 or 3: X1) Protein markers; X2) fluorescence imaging; X3) Photoelectric correlation microscopy; X4) Track the structure and / or morphology of proteins, subcellular organelles, localized cellular regions, cells, or small animal embryos; X5) Analyze the structure and / or location of the target protein; X6) Prepare protein-labeled products; X7) Prepare fluorescence imaging products; X8) Prepare photoelectric correlation microscopic imaging products; X9) Prepare products that track the structure and / or morphology of proteins, subcellular organelles, localized cellular regions, cells, or small animal embryos; X10) Prepare products for analyzing the structure and / or localization of the target protein; X11) Prepare relevant viruses or transgenic animals, label specific proteins, specific cells or specific organelles to achieve rapid transparent tissue imaging and expanded super-resolution imaging.

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