Three-fragment fluorescence complementation system based on fluorescent protein mEosEM and application of three-fragment fluorescence complementation system

Abstract

The invention discloses a fluorescent protein mEosEM-based three-fragment fluorescence complementation system and application thereof. The three-fragment fluorescence complementation system comprises a first carrier, a second carrier and a third carrier which respectively contain nucleotide sequences for expressing first, second and third target proteins; the first target protein, the second target protein and the third target protein are fusion proteins containing AmEosEM, BmEosEM and CmEosEM respectively, and the AmEosEM, the BmEosEM and the CmEosEM are three protein fragments split from the n / n + 1 site and the m / m + 1 site of mEosEM respectively. When the three protein fragments are respectively connected with the three proteins capable of interacting, the three fluorescent protein fragments form a complete structure and emit a fluorescent signal. The three-fragment fluorescence complementation system can be applied to imaging application of interaction between proteins to research the interaction between the proteins.

Description

Technical Field

[0001] This application belongs to the technical field of fluorescence imaging of protein interactions, and particularly relates to a three-fragment fluorescence complementation system based on the fluorescent protein mEosEM and its application. Background Art

[0002] Imaging of protein interactions is a visualization technique for studying the interaction relationships between proteins. Proteins rarely function alone in cells, but rather execute their biological functions by interacting with other proteins. Protein interaction is a crucial process in biology. Protein complexes can precisely regulate various reactions in cells, such as DNA replication, gene expression regulation, metabolic pathways, cell cycle control, and signal transduction, etc., ensuring the orderly progress of life activities. Therefore, using protein interaction imaging technology can observe these interactions in real time in the natural environment of cells or tissues, which helps to deeply understand the functions of proteins and the signal transduction mechanisms in cells.

[0003] Among them, bimolecular fluorescence complementation (BiFC) technology is a relatively mature protein interaction imaging technology. However, due to the problem of easy generation of false positive signals in this technology, a more sensitive, more specific and wider applicable three-molecule fluorescence complementation technology has been further derived on this basis. The three-molecule fluorescence complementation imaging technology (TFFC) splits a complete fluorescent protein into three non-fluorescent fragments. These three fragments cannot spontaneously complement and restore fluorescence without the action of interacting proteins. If three interacting proteins are respectively fused with the three fragments of the split fluorescent protein, under the action of these three interacting proteins, the three fragments of the split fluorescent protein will approach each other, thus restoring the conformation of the complete fluorescent protein and emitting specific fluorescence. Currently, three-fragment fluorescence complementation systems based on mIrisFP, Venus, etc. have been constructed in existing research.

[0004] mEosEM is a photo-convertible fluorescent protein that can achieve super-resolution optoelectronic correlation imaging (Zhifei Fu et al. Nature methods. 2019). It has been widely used in the field of super-resolution microscopy, such as photoactivated localization microscopy (PALM). In the initial state, the peak excitation wavelength of mEosEM is at 506 nm, and the peak emission wavelength is at 516 nm, showing green fluorescence. When activated by 405 nm ultraviolet light, the His62-Tyr63-Gly64 peptide bond inside the chromophore of mEosEM will break and recombine, resulting in a red shift of its peak excitation wavelength and peak emission wavelength to 571 nm and 581 nm respectively, thus turning into red fluorescence. In addition, after activation, the photobleaching tolerance of the red fluorescence emission of mEosEM is significantly enhanced, and it can support long-term dynamic tracking for more than 10 minutes. The red fluorescent state of mEosEM has an equilibrium quantum yield (0.55) and extinction coefficient (37,000 M -1 cm -1 )), and this property ensures that high signal-to-noise ratio can be obtained during imaging.

[0005] However, there is currently no three-fragment fluorescence complementation system based on mEosEM. Given the significant advantages of mEosEM in fluorescence imaging applications, it is very necessary to develop the corresponding three-fragment fluorescence complementation system. Summary of the Invention

[0006] In view of this, the primary object of this application is to provide a three-fragment fluorescence complementation system based on the fluorescent protein mEosEM. Taking the fluorescent protein mEosEM as a template, it is split into three non-fluorescent protein fragments, AmEosEM, BmEosEM, and CmEosEM. These three protein fragments themselves do not have the properties of the full-length protein. When these three fluorescent protein fragments are respectively linked to the first, second, and third proteins to be tested, if the first, second, and third proteins to be tested can interact with each other to spontaneously form a trimer or complex, then the three fluorescent protein fragments can be fused and expressed with each other, enabling the three protein fragments to form a complete structure and emit a fluorescent signal.

[0007] To achieve the above object, this application adopts the following technical solutions: One aspect of this application provides a three-fragment fluorescence complementation system based on the fluorescent protein mEosEM, including a first vector, a second vector, and a third vector. The first, second, and third vectors respectively contain nucleotide sequences for expressing the first, second, and third target proteins; the first target protein is a fusion protein of the first protein to be tested and AmEosEM, the second target protein is a fusion protein of the second protein to be tested and BmEosEM, and the third target protein is a fusion protein of the third protein to be tested and CmEosEM; The AmEosEM, BmEosEM, and CmEosEM are respectively three protein fragments split from the fluorescent protein mEosEM; the AmEosEM is a protein fragment composed of amino acids at positions 1 to n of the fluorescent protein mEosEM, the BmEosEM is a protein fragment composed of amino acids at positions n + 1 to m of the fluorescent protein mEosEM, and the CmEosEM is a protein fragment composed of amino acids at positions m + 1 to 227 of the fluorescent protein mEosEM; where n = 86 and m = 151; or, n = 151 and m = 186; or, n = 151 and m = 183.

[0008] Another aspect of the present application provides an imaging application of the three - fragment fluorescence complementation system based on the fluorescent protein mEosEM as described above in protein - protein interactions.

[0009] Advantages of the present application: Taking the fluorescent protein mEosEM as a template, the present application splits it into three non - fluorescent fragments AmEosEM, BmEosEM, and CmEosEM. The three protein fragments obtained by the splitting method in the present application do not have the properties of the full - length protein. When these three fluorescent protein fragments are respectively linked to the first, second, and third proteins to be detected, if the first, second, and third proteins to be detected can interact spontaneously to form a trimer or complex, the three fluorescent protein fragments can be fused and expressed with each other, and the three fluorescent protein fragments form a complete structure and emit a fluorescent signal.

[0010] The fluorescence complementation imaging system described above can verify its results through cell transfection at room temperature, and can be used to verify protein - protein interactions, especially specifically verify whether other proteins are trimeric complexes. This system can be used as a simple and efficient system for verifying trimeric interactions. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 It is a map of the expression vector used in the embodiments of the present application.

[0012] Figure 2 It is a schematic structural diagram of the splitting scheme of the fluorescent protein mEosEM in Embodiments 2 - 4 of the present application. The blue is the N - terminus and the red is the C - terminus. Among them, Figure 2 A in it is a schematic structural diagram of the fluorescent protein mEosEM, Figure 2 B in it is a schematic structural diagram of the splitting scheme in Embodiment 2, Figure 2 C in it is a schematic structural diagram of the splitting scheme in Embodiment 3, Figure 2 D in it is a schematic structural diagram of the splitting scheme in Embodiment 4.

[0013] Figure 3 Flow cytometry contour maps of each splitting scheme in Example 5 of the present application, wherein the negative control group ( Figure 3 A in Example 2 ( Figure 3 B), Example 3 ( Figure 3 C), Example 4 ( Figure 3 D in), Comparative Example 1 ( Figure 3 E in), Comparative Example 2 ( Figure 3 F in), positive control (Foldon+mEosEM) ( Figure 3 in G).

[0014] Figure 4 are flow cytometry histograms of each splitting scheme in Example 5 of the present application, wherein the negative control group ( Figure 4 A in Example 2 ( Figure 4 B), Example 3 ( Figure 4 C), Example 4 ( Figure 4 D in), Comparative Example 1 ( Figure 4 E in), Comparative Example 2 ( Figure 4 F in), positive control (Foldon+mEosEM) ( Figure 4 in G).

[0015] Figure 5 This is a bar graph of the cell transfection efficiency (i.e., the efficiency of forming a fluorescent protein complex) in Example 5 of the present application.

[0016] Figure 6 This is the experimental result of gel filtration chromatography in Example 6 of the present application, wherein: Figure 6 A in the figure is the gel filtration chromatography result after expression of mEosEM according to the splitting scheme in Example 4 when the first vector: the second vector: the third vector = 3:2:1; Figure 6 B in the figure is the gel filtration chromatography result after expression of mEosEM according to the splitting scheme in Example 4 when the first vector: the second vector: the third vector = 1:2:3; Figure 6 C in the figure is the gel filtration chromatography result after expression of mEosEM according to the splitting scheme in Example 2, where the first vector: the second vector: the third vector = 2:3:1.

[0017] Figure 7 This is the SDS-PAGE electrophoresis detection result in Example 6 of the present application, wherein, Figure 7 A in the figure is the result of electrophoresis detection by SDS-PAGE after expression and purification of mEosEM according to the splitting scheme in Example 4, when the first vector: the second vector: the third vector = 3:2:1; Figure 7B in it is the result of SDS-PAGE electrophoresis detection after the expression and purification of mEosEM when the first vector: the second vector: the third vector = 1:2:3 according to the splitting scheme in Example 4; Figure 7 C in it is the result of SDS-PAGE electrophoresis detection after the expression and purification of mEosEM when the first vector: the second vector: the third vector = 2:3:1 according to the splitting scheme in Example 2. Detailed implementation manners

[0018] The implementation manners of the present application will be clearly and completely described below. The technical solutions in the following described implementation manners are exemplary and only possible technical implementations of the present application, not all possible implementations. Those skilled in the art can fully combine the implementation manners of the present application to obtain other implementation manners without creative labor, and these implementation manners are also within the protection scope of the present application.

[0019] The first aspect of the present application discloses a three-fragment fluorescence complementation system based on the fluorescent protein mEosEM. The three-fragment fluorescence complementation system includes a first vector, a second vector, and a third vector. The first, second, and third vectors respectively contain nucleotide sequences for expressing the first, second, and third target proteins; the first target protein is a fusion protein of a first protein to be detected and AmEosEM, the second target protein is a fusion protein of a second protein to be detected and BmEosEM, and the third target protein is a fusion protein of a third protein to be detected and CmEosEM; AmEosEM, BmEosEM, and CmEosEM are respectively three protein fragments split from the fluorescent protein mEosEM.

[0020] If the first protein to be detected, the second protein to be detected, and the third protein to be detected can interact with each other, they can spontaneously form a trimer or complex in the cell, so that the three fluorescent protein fragments AmEosEM, BmEosEM, and CmEosEM that do not emit light by themselves approach and fuse with each other, reorganize into a complete structure and emit a fluorescence signal. Therefore, the interaction between known proteins or unknown proteins can be verified through this three-fragment fluorescence complementation system, which is particularly suitable for specifically verifying the interaction of trimeric proteins.

[0021] Those skilled in the art know that the fluorescent protein mEosEM is a protein composed of 227 amino acids, and its full-length amino acid sequence is as follows: MVSAIKPDMRIKLRMEGNVNGHHFVIDGEGTGKPYEGKQTMDLEVKEGGPLPFAFDILTTAFHYGNRVFVKYPDNIQDYFKQSFPKGYSWERSMTFEDGGICNARNDITMEGDTFYNKVRFYGTNFPANGPVMQKKTLKWEPSTEKMYVRDGVLTGDIEMALLLEGGAHYRCDFRTTYKAKEKGVKLPGAHFVDHAIEILSHDKDYNKVKLYEHAVAHSGLPDNARR (SEQ ID NO.1).

[0022] The full-length nucleotide sequence of the fluorescent protein mEosEM in this application is as follows: ATGGTGTCCGCCATCAAGCCCGATATGCGGATCAAGCTGAGAATGGAAGGCAACGTGAACGGCCACCACTTCGTTATCGACGGCGAGGGCACCGGCAAACCTTACGAGGGCAAGCAGACCATGGACCTGGAAGTGAAAGAGGGCGGACCTCTGCCTTTCGCCTTTGATATTCTGACAACCGCCTTCCACTACGGCAATAGAGTGTTCGTGAAGTACCCAGACAACATCCAGGACTACTTCAAGCAAAGCTTCCCCAAGGGATACAGCTGGGAGAGAAGCATGACCTTCGAGGACGGAGGCATCTGTAATGCTAGAAACGACATCACCATGGAAGGCGACACCTTTTATAACAAGGTCCGGTTCTACGGCACAAACTTCCCTGCTAACGGCCCTGTGATGCAGAAAAAGACCCTGAAGTGGGAACCTTCTACAGAGAAGATGTACGTGCGCGACGGCGTGCTGACAGGCGATATCGAGATGGCCCTGCTGCTGGAAGGAGGAGCTCATTATAGATGCGACTTCCGGACCACCTACAAGGCCAAGGAAAAGGGCGTGAAGCTCCCCGGCGCCCACTTTGTGGACCACGCCATCGAGATCCTGAGCCACGACAAGGATTACAACAAGGTGAAACTGTACGAGCACGCCGTGGCCCACAGCGGCCTGCCAGATAACGCCAGAAGA (SEQ ID NO.2).

[0023] In this application, the fluorescent protein mEosEM is split into three non-fluorescent protein fragments, AmEosEM, BmEosEM, and CmEosEM. When these three protein fragments are respectively fused with three interacting proteins and co-expressed in the same cell, the three non-fluorescent protein fragments can be brought closer by the interacting proteins and restored to the complete conformation of the fluorescent protein mEosEM, thereby exciting the generation of a fluorescent signal. Therefore, this three-fragment fluorescence complementation system can study the interaction of multiple proteins through fluorescence imaging reactions under physiological temperature conditions of cells.

[0024] In this application, there are no particular limitations on the first protein to be tested, the second protein to be tested, and the third protein to be tested. They can be any proteins in the art that have interactions and can form trimers or complexes, or any proteins that do not have interactions. When connecting and fusing the first protein to be tested, the second protein to be tested, and the third protein to be tested with the split fluorescent protein fragments respectively to construct the corresponding vectors, when designing primers, a linker sequence or an adapter sequence can be added as needed between the first / second / third protein to be tested and the split fluorescent protein fragments, and corresponding designs can be made according to actual needs. Those skilled in the art have such capabilities, so there are no particular limitations. When these fusion proteins are co-expressed in the same cell, if the first protein to be tested, the second protein to be tested, and the third protein to be tested have interactions, the split fluorescent protein fragments will approach each other, restore the conformation of the complete fluorescent protein, and then emit specific fluorescence; if the first protein to be tested, the second protein to be tested, and the third protein to be tested do not have interactions, the split fluorescent protein fragments cannot be brought closer to each other, and thus the conformation of the complete fluorescent protein cannot be restored, and no fluorescence will be emitted. Therefore, based on the three-fragment fluorescence complementation system described in this application, it is possible to study whether there are interactions between known proteins or unknown proteins.

[0025] In some examples, the first protein to be tested, the second protein to be tested, and the third protein to be tested are trimeric proteins. For example, they can be heterotrimeric monomers or homotrimeric proteins.

[0026] In some preferred examples, the trimeric protein is a homotrimeric protein, and the homotrimeric protein is Foldon.

[0027] Those skilled in the art know that the trimeric protein Foldon is a natural trimeric domain derived from the T4 phage fiber protein and consists of a β-propeller conformation. Foldon is an efficient trimerization tool. The amino acid sequence of the trimeric protein Foldon in this application is as follows: GYIPEAPRDGQAYVRKDGEWVLLSTFLGRS (SEQ ID NO.3).

[0028] In this application, the full-length nucleotide sequence of the trimeric protein Foldon is as follows: ATGGCAGGCTACATTCCTGAAGCCCCTAGAGACGGACAGGCCTATGTCAGAAAGGACGGCGAGTGGGTGCTGCTGAGCACCTTCCTGGGCAGAAGCGGCAGAAGC (SEQ ID NO.4).

[0029] In a preferred example of this application, taking the trimeric protein Foldon as an example, Foldon is respectively fused with three protein fragments obtained by splitting the fluorescent protein mEosEM. When these fusion proteins are co-expressed in the same cell, under the trimerization of Foldon, the three split protein fragments will approach each other, restore the conformation of the complete fluorescent protein, and then emit specific fluorescence.

[0030] In this application, the AmEosEM is a protein fragment composed of amino acids at positions 1 to n of the fluorescent protein mEosEM, the BmEosEM is a protein fragment composed of amino acids at positions n + 1 to m of the fluorescent protein mEosEM, and the CmEosEM is a protein fragment composed of amino acids at positions m + 1 to 227 of the fluorescent protein mEosEM; where n = 86 and m = 151; or, n = 151 and m = 186; or, n = 151 and m = 183.

[0031] It can be seen that in this application, mEosEM can have three splitting schemes, and each splitting scheme can achieve the expected effect of this application.

[0032] In some examples, when n = 86 and m = 151, the first vector contains the nucleotide sequence SEQ ID NO.5 for expressing the protein fragment AmEosEM; the second vector contains the nucleotide sequence SEQ ID NO.6 for expressing the protein fragment BmEosEM; the third vector contains the nucleotide sequence SEQ ID NO.7 for expressing the protein fragment CmEosEM.

[0033] In some other examples, when n = 151 and m = 186, the first vector contains the nucleotide sequence SEQ ID NO.8 for expressing the protein fragment AmEosEM; the second vector contains the nucleotide sequence SEQ ID NO.9 for expressing the protein fragment BmEosEM; the third vector contains the nucleotide sequence SEQ ID NO.10 for expressing the protein fragment CmEosEM as shown.

[0034] In some other examples, when n = 151 and m = 183, the first vector contains the nucleotide sequence SEQ ID NO.8 for expressing the protein fragment AmEosEM; the second vector contains the nucleotide sequence SEQ ID NO.11 for expressing the protein fragment BmEosEM; the third vector contains the nucleotide sequence SEQ ID NO.12 for expressing the protein fragment CmEosEM.

[0035] Furthermore, it can be understood that the first, second, and third vectors in the three-fragment fluorescence complementation system are obtained by inserting the nucleotide sequences for expressing the first, second, and third target proteins into the corresponding expression vectors. In some examples, the nucleotide sequences for expressing the first, second, and third target proteins are obtained by operably linking the nucleotide sequences for expressing the first / second / third proteins to be tested with the nucleotide sequences for expressing the protein fragments AmEosEM / BmEosEM / CmEosEM through a linker sequence. Among them, the linker sequence can be specifically designed according to the types of the first / second / third proteins to be tested, without specific limitations. In some examples of this application, the first / second / third proteins to be tested are all Foldon, and the linker sequence used is GGSGG, and its nucleotide sequence is GGCGGATCCGGAGGC (SEQ ID NO.13), but it is not limited thereto.

[0036] It can be understood that in this application, the "operably linked (connected)" or "operationally connected (connected)" refers to the functional spatial arrangement of two or more nucleic acid regions or nucleic acid sequences.

[0037] In this application, the expression vector is a genetically engineered vector used for highly expressing foreign genes in host cells and producing corresponding proteins. It is based on a cloning vector and has additional expression regulatory elements (such as promoters, ribosome binding sites, terminators, etc.), enabling the target gene to be transcribed and translated. In this application, the expression vector can be a prokaryotic expression vector or a eukaryotic expression vector. Among them, the prokaryotic expression vector is a type of vector suitable for bacteria (such as Escherichia coli), with prokaryotic promoters (such as T7, lac) and SD sequences. Specific examples include the pET series. The eukaryotic expression vector is a type of vector suitable for yeast, mammalian, or insect cells, containing eukaryotic promoters (such as CMV) and secretion signal peptides. In this application, it is preferably a mammalian cell expression vector, and specific examples include pcDNA3.1, etc. In some specific examples of this application, the expression vector used is PM0147-pIHM_Fc_HRV3C_mEOS constructed by the applicant's laboratory (see the Chinese patent application with the publication number CN119799784A).

[0038] In this application, there is no special limitation on the construction method of the three-fragment fluorescence complementation system, and conventional methods in the art can be used. In some examples of this application, first, the nucleotide sequence of the Foldon-mEosEM fragment (SEQ ID NO.14) is obtained by polymerase chain reaction (PCR); then the obtained Foldon-mEosEM gene fragment is inserted into the multiple cloning site of the PM0147-pIHM_Fc_HRV3C_mEOS vector to construct the vector Foldon-mEosEM.

[0039] In addition, it can be understood that some specific protein tags can be operably linked according to needs during the specific design. Specific examples include, but are not limited to, purification tags, etc. For example, in some examples, a his tag (GGCAGCGGACACCACCACCACCACCAC) is added.

[0040] Furthermore, by designing corresponding primers, the nucleotide sequences for expressing the first target protein, the second target protein, and the third target protein are respectively obtained by PCR amplification from the vector Foldon-mEosEM, and then inserted into the multiple cloning site of the Foldon-mEosEM vector to construct the first vector, the second vector, and the third vector.

[0041] Among them, those skilled in the art can design the primer and select the multiple cloning site accordingly according to the inserted protein fragment. Those skilled in the art have such capabilities and will not be elaborated here specifically.

[0042] The second aspect of the present application discloses the imaging application of the three-fragment fluorescence complementation system based on the fluorescent protein mEosEM in protein-protein interactions as described above.

[0043] In specific applications, the first vector, the second vector, and the third vector constructed in the previous text are co-transfected into the cells to be detected according to a certain ratio. After culturing for a period of time, fluorescence imaging is performed for observation. If fluorescence can be observed, it indicates that the first / second / third protein to be detected has an interaction and can form a trimer or complex; if fluorescence cannot be observed, it indicates that the first / second / third protein to be detected does not have an interaction. Among them, the ratio of the first vector, the second vector, and the third vector is not particularly limited, and those skilled in the art can determine it through experimental methods. For example, in some examples, the mass ratio of the first vector, the second vector, and the third vector is preferably (1-5):(1-5):(1-5).

[0044] The following are specific examples of the present application. It should be noted that the following specific examples are only for illustrative purposes and do not limit the scope of the present application in any way.

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this application belongs. The terms used in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0046] In addition, unless otherwise specified, the methods without specific conditions or steps recorded are conventional methods, and the reagents and materials used can be obtained from commercial sources.

[0047] In the following examples, three sets of three-fragment fluorescence complementation systems based on the fluorescent protein mEosEM were constructed using the trimeric protein Foldon as an example to verify that the three-fragment splitting scheme of the fluorescent protein mEosEM in this application is feasible and successful.

[0048] Example 1 Construction of Foldon-mEosEM Vector 1. PM0147-pIHM_Fc_HRV3C_mEOS expression vector The PM0147-pIHM_Fc_HRV3C_mEOS expression vector (6731 bp in length) used in this example was constructed by our laboratory. For details, see the Chinese patent application with the publication number CN119799784A, and the map is shown in Figure 1 .

[0049] 2. After digesting the PM0147-pIHM_Fc_HRV3C_mEOS expression vector with the restriction enzymes BamH I and Bgl II, insert the nucleotide sequence of the Foldon-mEosEM fragment into it to obtain a vector. The specific steps are as follows: (1) Obtain the gene sequence of the Foldon-mEosEM fragment (SEQ ID NO.14) by PCR amplification. The primer information involved is shown in Table 1: Table 1 Primer and its sequence information

[0050] (2) Use the double restriction enzyme sites BamH I and Bgl II to insert the Foldon-mEosEM gene fragment obtained in step (1) into the multiple cloning site of the PM0147-pIHM_Fc_HRV3C_mEOS vector to construct the plasmid Foldon-mEosEM.

[0051] Example 2 Construction of a three-fragment fluorescence complementation system (n = 86 and m = 151) In this example, a three-fragment fluorescence complementation system based on the fluorescent protein mEosEM was constructed, including a first vector, a second vector, and a third vector respectively. The splitting sites of mEosEM are located at positions 86 / 87 and 151 / 152 of mEosEM, resulting in a protein fragment - AmEosEM composed of amino acids at positions 1 to 86 of the fluorescent protein mEosEM, a protein fragment - BmEosEM composed of amino acids at positions 87 to 151 of the fluorescent protein mEosEM, and a protein fragment - CmEosEM composed of amino acids at positions 152 to 227 of the fluorescent protein mEosEM. The specific steps are as follows: (1) Construction of the first vector: On the plasmid Foldon-mEosEM constructed in Example 1, obtain the nucleotide sequence for expressing the first target protein by PCR amplification, that is, Foldon-linker-AmEosEM. Among them, the nucleotide sequence for expressing Foldon is as shown in SEQ ID NO.4, the nucleotide sequence of linker is as shown in SEQ ID NO.13, and the nucleotide sequence for expressing the protein fragment AmEosEM is as shown in SEQ ID NO.5. The primer information involved is shown in Table 2: Table 2 Primer and its sequence information

[0052] The nucleotide sequence for expressing the first target protein was inserted into the multiple cloning site of plasmid Foldon-mEosEM using the double restriction enzyme sites BamH I and Bgl II to construct the first vector.

[0053] (2)Construction of the second vector: The nucleotide sequence for expressing the second target protein, i.e., Foldon-linker-BmEosEM, was obtained by PCR amplification on the plasmid Foldon-mEosEM constructed in Example 1. Among them, the nucleotide sequence of the expressed protein fragment BmEosEM is shown in SEQ ID NO.6, and the primer information involved is shown in Table 3: Table 3 Primer and its sequence information

[0054] The nucleotide sequence for expressing the second target protein was inserted into the multiple cloning site of plasmid Foldon-mEosEM using the double restriction enzyme sites BamH I and Bgl II to construct the second vector.

[0055] (3)Construction of the third vector: The nucleotide sequence for expressing the third target protein, i.e., Foldon-linker-CmEosEM, was obtained by PCR amplification on the plasmid Foldon-mEosEM constructed in Example 1. Among them, the nucleotide sequence of the expressed protein fragment CmEosEM is shown in SEQ ID NO.7, and the primer information involved is shown in Table 4: Table 4 Primer and its sequence information

[0056] The nucleotide sequence for expressing the third target protein was inserted into the multiple cloning site of plasmid Foldon-mEosEM using the double restriction enzyme sites BamH I and Bgl II to construct the third vector.

[0057] Example 3 Construction of a three-fragment fluorescence complementation system (n = 151 and m = 186) In this embodiment, another three-fragment fluorescence complementation system based on the fluorescent protein mEosEM was constructed, which respectively included a first vector, a second vector, and a third vector. The splitting sites of mEosEM were located at positions 151 / 152 and 186 / 187 of mEosEM, resulting in a protein fragment - AmEosEM composed of amino acids at positions 1 to 151 of the fluorescent protein mEosEM, a protein fragment - BmEosEM composed of amino acids at positions 152 to 186 of the fluorescent protein mEosEM, and a protein fragment - CmEosEM composed of amino acids at positions 187 to 227 of the fluorescent protein mEosEM. The specific steps are as follows: (1) Construction of the first vector: The nucleotide sequence for expressing the first target protein, namely Foldon-linker-AmEosEM, was obtained by PCR amplification on the plasmid Foldon-mEosEM constructed in Example 1. Among them, the nucleotide sequence for expressing the protein fragment AmEosEM is shown in SEQ ID NO.8, and the primer information involved is shown in Table 5: Table 5 Primer and its sequence information

[0058] The nucleotide sequence for expressing the first target protein was inserted into the multiple cloning site of the plasmid Foldon-mEosEM using the double restriction enzyme sites BamH I and Bgl II to construct the first vector.

[0059] (2) Construction of the second vector: The nucleotide sequence for expressing the second target protein, namely Foldon-linker-BmEosEM, was obtained by PCR amplification on the plasmid Foldon-mEosEM constructed in Example 1. The nucleotide sequence of the protein fragment BmEosEM is shown in SEQ ID NO.9, and the primer information involved is shown in Table 6: Table 6 Primer and its sequence information

[0060] The nucleotide sequence for expressing the second target protein was inserted into the multiple cloning site of the plasmid Foldon-mEosEM using the double restriction enzyme sites BamH I and Bgl II to construct the second vector.

[0061] (3) Construction of the third vector: The nucleotide sequence for expressing the third target protein, i.e., Foldon-linker-CmEosEM, was obtained by PCR amplification on the plasmid Foldon-mEosEM constructed in Example 1. Among them, the nucleotide sequence of the expressed protein fragment CmEosEM is shown in SEQ ID NO.10, and the primer information involved is shown in Table 7: Table 7 Primers and Their Sequence Information

[0062] The nucleotide sequence for expressing the third target protein was inserted into the multiple cloning site of the plasmid Foldon-mEosEM using the double restriction enzyme sites BamH I and Bgl II to construct the third vector.

[0063] Example 4 Construction of a Three-Fragment Fluorescence Complementation System (n = 151 and m = 183) In this example, another three-fragment fluorescence complementation system based on the fluorescent protein mEosEM was constructed, including a first vector, a second vector, and a third vector respectively. The splitting sites of mEosEM are located at positions 151 / 152 and 183 / 184 of mEosEM, resulting in a protein fragment - AmEosEM composed of amino acids at positions 1 to 151 of the fluorescent protein mEosEM, a protein fragment - BmEosEM composed of amino acids at positions 152 to 183 of the fluorescent protein mEosEM, and a protein fragment - CmEosEM composed of amino acids at positions 184 to 227 of the fluorescent protein mEosEM. The specific steps are as follows: (1) Construction of the first vector: Refer to the construction of the first vector in Example 3.

[0064] (2) Construction of the second vector: The nucleotide sequence for expressing the second target protein, i.e., Foldon-linker-BmEosEM, was obtained by PCR amplification on the plasmid Foldon-mEosEM constructed in Example 1. Among them, the nucleotide sequence of the expressed protein fragment BmEosEM is shown in SEQ ID NO.11, and the primer information involved is shown in Table 8: Table 8 Primers and Their Sequence Information

[0065] The nucleotide sequence for expressing the second target protein was inserted into the multiple cloning site of the plasmid Foldon-mEosEM using the double restriction enzyme sites BamH I and Bgl II to construct the second vector.

[0066] (3) Construction of the third vector: The nucleotide sequence for expressing the third target protein was obtained by PCR amplification on the plasmid Foldon-mEosEM constructed in Example 1, namely Foldon-linker-CmEosEM. Among them, the nucleotide sequence of the expressed protein fragment CmEosEM is shown in SEQ ID NO. 12, and the primer information involved is shown in Table 9: Table 9 Primers and Their Sequence Information

[0067] The nucleotide sequence for expressing the third target protein was inserted into the multiple cloning site of the plasmid Foldon-mEosEM using the double restriction enzyme sites BamH I and Bgl II to construct the third vector.

[0068] Comparative Example 1 In this comparative example, the split sites of the fluorescent protein Venus disclosed in the Chinese patent application with the publication number CN109385450A were split in mEosEM. The split sites are located at positions 149 / 150 and 165 / 166 of mEosEM, and the corresponding vector was prepared with reference to the method in Example 3.

[0069] Among them, the primer information involved in the process of constructing the vector is shown in Table 10: Table 10 Primers and Their Sequence Information

[0070] Comparative Example 2 In this comparative example, the split sites of GFP disclosed in the Chinese patent application with the publication number CN109735569A were split in mEosEM. The split sites are located at positions 185 / 186 and 204 / 205 of mEosEM, and the corresponding vector was prepared with reference to the method in Example 3.

[0071] Among them, the primer information involved in the process of constructing the vector is shown in Table 11: Table 11 Primers and Their Sequence Information

[0072] Example 5 Identification of Green Fluorescent Protein Recombination 1. 293T Cell Culture The 293T cells (CRL-11268 ™ , ACTT) were cultured in complete DMEM medium (C11885500BT, Gibico) containing 10% fetal bovine serum, 1% GlutaMax, non-essential amino acids, sodium pyruvate, ampicillin, and streptomycin in 5% CO 2Cultured in a 37°C constant temperature cell incubator, the culture medium was replaced and subcultured according to the cell growth status.

[0073] The source of the components in the culture medium is shown in Table 12: Table 12 Information on the components of the culture medium

[0074] The specific steps are as follows: Carefully aspirate the culture medium, wash the cells with PBS, add 1 mL of trypsin, and incubate in a 37°C constant temperature cell incubator for 1 min until the cells are completely detached from the wall; add 9 mL of DMEM medium, pipette to mix the cells evenly, take 1 mL and transfer it to a new culture dish, and add 9 mL of fresh medium to continue the culture.

[0075] 2. Transfection of 293T cells One day before the transfection experiment, 5×10 6 293T cells were suspended in 10 mL of complete medium and cultured overnight in a cell incubator at 37°C and 5% CO 2 to reach a cell confluence of 70%-80%. Before transfection, the cells were digested normally, 10 mL of complete medium was added to disperse the digested cells, the cells were suspended and dispersed, and the cells were counted. Plating was carried out at 2.5 mL per well in a 6-well plate and 0.67×10 6 cells per well.

[0076] 0.3 μg each of the first vector, the second vector, and the third vector in Example 2 were respectively added to a centrifuge tube containing 100 μl of Opti-MEM medium, and transfection liposome reagent was added (the mass-volume ratio of the plasmid to the transfection liposome reagent was 1:1). The incubated plasmid-transfection reagent was evenly dispersed and added to the 293T cells, and cultured in a cell incubator at 37°C and 5% CO 2 for 48 h.

[0077] The first vector, the second vector, and the third vector in Example 3, Example 4, Comparative Example 1, and Comparative Example 2 were all transfected using the same method as above.

[0078] At the same time, a negative control (blank) and a positive control (transfection with plasmid Foldon-mEosEM) were set up. Among them, the transfection with plasmid Foldon-mEosEM was carried out using the same method as above.

[0079] 3. Sample preparation for flow cytometry (1) Prepare FACS solution: Add 1 mL of FBS solution to PBS buffer and make up the volume to 50 mL.

[0080] (2) Remove the cells from the incubator, aspirate and discard the supernatant medium on top of the cells. Add 1 mL of FACS solution to each well of the plasmid transfection wells, and add 1 mL of FACS solution to the control group.

[0081] (3) Use a pipette to disperse the cells, aliquot the cells from each well into flow tubes, 1 mL per tube, and centrifuge at 2000 rpm at 4 °C for 6 min.

[0082] (4) After centrifugation, discard the supernatant. Add 500 μl of FACS solution to each tube to resuspend the cells; centrifuge at 2000 rpm at 4 °C for 6 min.

[0083] (5) Prepare 2% fixing solution during centrifugation: Add 5 mL of 4% tissue cell fixing solution to 5 mL of 1×PBS solution, and vortex to mix evenly.

[0084] (6) After centrifugation, discard the supernatant. Add 500 μl of 2% fixing solution to each tube, vortex to mix evenly, and wait for detection by flow cytometry.

[0085] Among them, Figure 3 and Figure 4 respectively show the test results of the three-fragment fluorescence complementation system in Examples 2-4 and Comparative Examples 1 and 2. If a functional fluorescence is formed, a fluorescence signal can be seen in FITC. If the splitting is not successful, no fluorescence signal can be seen. Through Figure 3 and Figure 4 it can be seen that fluorescence signals can be seen in FITC for Examples 2, 3, and 4 and the positive control, while no fluorescence signals can be seen in FITC for Comparative Examples 1 and 2 and the negative control, indicating that the splitting scheme in the present application is feasible and successful.

[0086] Further, the cell transfection efficiency graph as shown in Figure 5 is obtained by counting the percentage of FITC fluorescence-positive cells among all cells. It can be seen that the transfection efficiencies of Examples 2-4 are 24.2%, 35%, and 33.5% respectively; while the transfection efficiencies of Comparative Examples 1 and 2 are 0.51% and 0.54% respectively. This shows that the splitting sites in Comparative Examples 1 and 2 cannot enable mEosEM to form a complete structure and function.

[0087] Example 6 Protein Purification 1. Solution Preparation (1) Preparation of cell lysis solution (100 mL): 4 ml of 500 mM Imidazole Buffer + 8.18 g of NaCl + 5 ml of 10% V / W NP-40 + 10 ml of 5% V / W Sodium deoxycholate + 0.736 g of Glu (glutamic acid) + 0.84 g of Arg (arginine).

[0088] (2)Imidazole gradient eluent: Prepare low-concentration imidazole eluents with concentrations of 20 mM and 50 mM respectively using 500 mM Imidazole Buffer and PBS, and adjust the pH to 7.6.

[0089] 2. Transfection of 293F cells (FreeStyle™ 293-F cells, product number R79007, Gibco) (1)Cell counting: Count using ordinary bright field / Trypan blue staining (add 25 μL to a glass slide).

[0090] (2)Cell dilution: Dilute the cells to a transfection concentration of 1×10 6 cells / mL (floating range of 0.8 - 1×10 6 cells / mL).

[0091] (3)Plasmid filtration: Add the plasmid to be transfected (1 mg plasmid corresponds to 1 L of cells (concentration 1×10 6 cells / mL)) to 1 mL of OptiPRO™ SFM (GIBCO, product number 12309019) medium, mix well, and filter and sterilize the plasmid-containing Optim medium using a 2.5 mL syringe + 0.22 μm filter membrane.

[0092] (4)Plasmid packaging: Slowly add the transfection reagent (LiFect293™ Transfection Reagent, LIFESCT Cat.#: M0002-01) (1 μg plasmid corresponds to 1 μL of transfection reagent) to the filtered plasmid-containing Optim medium, invert and mix well, and then let it stand at room temperature for 5 min.

[0093] (5)Transfect the cells: Add all the incubated plasmids to the diluted cells (1×10 6 cells / mL).

[0094] (6)Harvest the cell supernatant: 6 - 7 days after successful transfection, centrifuge at 12000 rpm for 10 min to collect the cell supernatant, and filter it through a 0.22 μm filter for protein purification.

[0095] 3. Primary purification of the protein: Purification using a nickel column (1)Collect the 293F cells transfected with the three fragments of Foldon-mEOS plasmid (i.e., the first vector, the second vector, and the third vector), centrifuge at low temperature and high speed for 15 min, and discard the supernatant to obtain the precipitate sample.

[0096] (2)Suspend the precipitate using 10 times the volume of cell lysate and lyse at 4°C for 60 min.

[0097] (3) Centrifuge the lysed cells at low temperature and high speed, aspirate the supernatant and filter it by suction.

[0098] (4) Add the pretreated nickel column gel to the supernatant filtrate by suction, incubate at 4 °C on a shaker at 130 rpm for 1 h.

[0099] (5) Transfer the binding liquid to a gravity column, wait for the liquid to drain, and collect the flow-through.

[0100] (6) Elute with an imidazole gradient eluent: ① Take 30 mL of an eluent of 20 mM IMD with PBS, pH 7.6 for elution; ② After the liquid has drained, take 30 mL of an eluent of 50 mM IMD with PBS, pH 7.6 for elution. ③ After the liquid has drained, take 30 mL of an eluent of 500 mM IMD with PBS, pH 7.6 for further elution.

[0101] (7) Collect the flow-through and the imidazole eluents of different concentrations in step (6), transfer the eluents to a concentrator tube respectively, and centrifuge and ultrafilter at 3500 rpm at low temperature.

[0102] (8) Wait until the eluent is concentrated to a volume of 1 mL, observe whether there is fluorescence in the solution, and perform the next purification step on the solution with observable fluorescence.

[0103] 4. Protein purification: Purify by gel filtration chromatography.

[0104] (1) Sterilization: Pass the protein solution concentrated to a volume of 1 mL through a 0.22 μm filter membrane and centrifuge at 14000 rpm at low temperature for 5 min.

[0105] (3) Equilibrate the system: Pre-equilibrate the SEC system, chromatography column and sample loop with PBS in advance.

[0106] (4) Inject the sample into the sample loop of the SEC system and start running the purification program.

[0107] (5) Sample the protein separated and purified according to the SEC result, perform SDS-PAGE electrophoresis detection, and concentrate and detect the concentration of the target sample.

[0108] 5. SDS-PAGE electrophoresis detection (1) Calculate the molecular weight of the target protein and predict the elution peak range of the target protein.

[0109] (2) Concentrate the protein solution in the target range according to the SEC chart.

[0110] (3) Sampling: Mix the protein concentrate with 5×loading buffer (sample loading buffer) in a volume ratio of 4:1.

[0111] (4) Heating: The protein concentrate mixed with the loading buffer is heated in a metal bath at 103 °C for 5 min to fully denature the protein.

[0112] (5) After centrifuging the sample at low speed and mixing it evenly, add it to the protein gel wells. After adding electrophoresis buffer to both the inner and outer parts of the electrophoresis tank, perform electrophoresis at a constant voltage of 140 V for 60 min.

[0113] (6) Peel off the protein gel that has completed electrophoresis and place it in the Fast Blue protein rapid staining solution for uniform coloring for 3 h.

[0114] (7) Place the stained protein gel in clear water and decolorize it for 60 min.

[0115] 6. Protein Yield Calculation (1) Transfer the target protein solution that has been purified and concentrated in two steps to an EP tube and vortex it evenly.

[0116] (2) Use a ultra-micro ultraviolet spectrophotometer, click on the A280 mode, input the theoretical extinction coefficient of the protein, and perform detection.

[0117] (3) Blank: Take 1 μL of PBS and drop it into the detector for blanking.

[0118] (4) Measurement: After the blanking is completed, wipe off the blank solution, take 1 μL of the protein solution and drop it into the detector, click on measurement, and record the concentration of the detected protein solution.

[0119] (5) Use a pipette to aspirate the volume of the detected protein solution multiple times, and calculate the final protein yield based on the volume and concentration of the protein solution.

[0120] Figure 6 The experimental results of gel filtration chromatography in this example are shown. Among them, Figure 6 A in is the result of gel filtration chromatography after the expression of mEosEM transfected with the splitting scheme in Example 4 (according to the mass ratio of the first vector, the second vector, and the third vector of 3:2:1). Figure 6 B in is the result of gel filtration chromatography after the expression of mEosEM transfected with the splitting scheme in Example 4 (according to the mass ratio of the first vector, the second vector, and the third vector of 1:2:3). Figure 6 C in is the result of gel filtration chromatography after the expression of mEosEM transfected with the splitting scheme in Example 2 (according to the mass ratio of the first vector, the second vector, and the third vector of 2:3:1).

[0121] It can be seen that after adopting the splitting scheme of the present application, the triple-split fluorescent protein can be purified and can be purified with other proteins through a molecular sieve chromatography gel, and further separation and purification can be achieved subsequently.

[0122] Figure 7 shows the SDS-PAGE electrophoresis detection results in this embodiment. Among them, Figure 7 A in is the electrophoresis detection result using SDS-PAGE after the expression and purification of mEosEM transfected with the splitting scheme in Example 4 (according to the mass ratio of the first vector, the second vector, and the third vector of 3:2:1). Figure 7 B in is the electrophoresis detection result using SDS-PAGE after the expression and purification of mEosEM transfected with the splitting scheme in Example 4 (according to the mass ratio of the first vector, the second vector, and the third vector of 1:2:3). Figure 7 C in is the electrophoresis detection result using SDS-PAGE after the expression and purification of mEosEM transfected with the splitting scheme in Example 2 (according to the mass ratio of the first vector, the second vector, and the third vector of 2:3:1).

[0123] It can be seen that after separation and purification, the band size and position meet the expectations.

[0124] It is proved by the above embodiments that the splitting scheme of the present application can successfully achieve in vitro recombinant expression through protein-protein interaction and can be obtained through expression and purification, indicating that the three splitting schemes of the fluorescent protein mEosEM in the present application are successful and feasible. This is of great significance for studying protein-protein interaction, especially for specifically verifying whether other proteins are trimeric complexes. This system can be used as a simple, efficient system for verifying trimeric interaction.

[0125] It should be noted that the present application is not limited to the above embodiments. The above embodiments are only examples, and embodiments with the same structure and the same function and effect as the technical idea within the technical solution scope of the present application are all included in the technical scope of the present application. In addition, within the scope not departing from the gist of the present application, various modifications that can be thought of by those skilled in the art to the embodiments and other ways constructed by combining some constituent elements of the embodiments are also included in the scope of the present application.

Claims

1. A three-fragment fluorescence complementation system based on the fluorescent protein mEosEM, characterized in that: It comprises a first vector, a second vector and a third vector, wherein the first, second and third vectors contain nucleotide sequences for expressing a first, second and third target protein, respectively; the first target protein is a fusion protein of a first protein to be tested and AmEosEM, the second target protein is a fusion protein of a second protein to be tested and BmEosEM, and the third target protein is a fusion protein of a third protein to be tested and CmEosEM; The AmEosEM, BmEosEM and CmEosEM are respectively three protein fragments separated from the fluorescent protein mEosEM whose amino acid sequence is shown in SEQ ID NO.1; the AmEosEM is a protein fragment composed of amino acids at positions 1 to n of the fluorescent protein mEosEM, the BmEosEM is a protein fragment composed of amino acids at positions n+1 to m of the fluorescent protein mEosEM, and the CmEosEM is a protein fragment composed of amino acids at positions m+1 to 227 of the fluorescent protein mEosEM; wherein n=86 and m=151; or, n=151 and m=186; or, n=151 and m=183.

2. The three-segment fluorescence complementation system based on the fluorescent protein mEosEM as claimed in claim 1, characterized in that: The nucleotide sequence of the fluorescent protein mEosEM is shown in SEQ ID NO.

2.

3. The three-fragment fluorescence complementation system based on the fluorescent protein mEosEM as claimed in claim 1, characterized in that: The first protein to be tested, the second protein to be tested and the third protein to be tested are trimeric proteins, and the trimeric protein is Foldon, and its amino acid sequence is shown in SEQ ID NO.

3.

4. The three-fragment fluorescence complementation system based on the fluorescent protein mEosEM as claimed in claim 3, characterized in that: The nucleotide sequence of Foldon is shown in SEQ ID NO.

4.

5. The three-fragment fluorescence complementation system based on the fluorescent protein mEosEM as claimed in claim 1, characterized in that: When n=86 and m=151, the first vector contains the nucleotide sequence SEQ ID NO.5 for expressing the protein fragment AmEosEM; the second vector contains the nucleotide sequence SEQ ID NO.6 for expressing the protein fragment BmEosEM; and the third vector contains the nucleotide sequence SEQ ID NO.7 for expressing the protein fragment CmEosEM.

6. The three-fragment fluorescence complementation system based on the fluorescent protein mEosEM as claimed in claim 1, characterized in that: When n=151 and m=186, the first vector contains the nucleotide sequence SEQ ID NO.8 for expressing the protein fragment AmEosEM; the second vector contains the nucleotide sequence SEQ ID NO.9 for expressing the protein fragment BmEosEM; and the third vector contains the nucleotide sequence SEQ ID NO.10 for expressing the protein fragment CmEosEM.

7. The three-segment fluorescence complementation system based on the fluorescent protein mEosEM as claimed in claim 1, characterized in that: When n=151 and m=183, the first vector contains the nucleotide sequence SEQ ID NO.8 for expressing the protein fragment AmEosEM; the second vector contains the nucleotide sequence SEQ ID NO.11 for expressing the protein fragment BmEosEM; and the third vector contains the nucleotide sequence SEQ ID NO.12 for expressing the protein fragment CmEosEM.

8. The three-segment fluorescence complementation system based on the fluorescent protein mEosEM according to any one of claims 1 to 7, characterized in that: The first, second and third vectors are obtained by inserting the nucleotide sequences for expressing the first, second and third target proteins into the corresponding expression vectors.

9. The three-segment fluorescence complementation system based on the fluorescent protein mEosEM as claimed in claim 8, characterized in that: The expression vector is a prokaryotic expression vector or a mammalian cell expression vector.

10. Application of the three-fragment fluorescence complementation system based on the fluorescent protein mEosEM as described in any one of claims 1 to 9 in imaging of protein-protein interactions.

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