A three-fragment fluorescence complementation system based on the fluorescent protein mEosEM and its applications

Abstract

The present application discloses a three-segment fluorescence complementation system based on the fluorescent protein mEosEM and its application. The three-segment fluorescence complementation system includes a first, a second, and a third vector containing nucleotide sequences for expressing the first, second, and third target proteins, respectively; the first, second, and third target proteins are fusion proteins containing AmEosEM, BmEosEM, and CmEosEM, respectively, and AmEosEM, BmEosEM, and CmEosEM are three protein fragments separated from the n / n+1 position and the m / m+1 position of mEosEM, respectively. When the three protein fragments are respectively connected to three proteins that can interact with each other, the three fluorescent protein fragments form a complete structure and emit a fluorescent signal. The three-segment fluorescence complementation system can be used in imaging applications of protein-protein interactions to study protein-protein interactions.

Description

Technical Field

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

[0002] Protein interaction imaging is a visualization technique used to study the interactions between proteins. Proteins rarely act alone within cells, but rather carry out their biological functions by interacting with other proteins. Protein interactions are a crucial process in biology. Protein complexes can precisely regulate a variety of intracellular reactions, such as DNA replication, gene expression regulation, metabolic pathways, cell cycle control, and signal transduction, ensuring the orderly progress of life activities. Therefore, protein interaction imaging technology can observe these interactions in real time in the natural environment of cells or tissues, providing a deeper understanding of protein functions and intracellular signaling mechanisms.

[0003] Among them, bimolecular fluorescence complementation (BiFC) is a relatively mature protein interaction imaging technique. However, due to its proneness to false positive signals, it has been further developed into trimolecular fluorescence complementation, which has higher sensitivity, better specificity, and a wider range of applications. Trimolecular fluorescence complementation (TFFC) involves splitting a complete fluorescent protein into three mutually non-fluorescent fragments. Without the interaction of interacting proteins, these three fragments cannot spontaneously complement each other and restore fluorescence. However, if three interacting proteins are fused to the three fragments of the split fluorescent protein, the three interacting proteins will bring the three fragments closer together, restoring the conformation of the complete fluorescent protein and emitting specific fluorescence. Currently, studies have constructed three-fragment fluorescence complementation systems based on mIrisFP, Venus, and other proteins.

[0004] mEosEM is a photoconvertible fluorescent protein that can achieve super-resolution photoelectrocorrelation imaging (Zhifei Fu et al. Nature methods. 2019). It has been widely used in super-resolution microscopic imaging techniques such as photoactivated localization microscopy (PALM). In its initial state, the excitation wavelength peak of mEosEM is at 506nm and the emission wavelength peak is at 516nm, exhibiting green fluorescence; when activated by 405nm ultraviolet light, the His62-Tyr63-Gly64 peptide bond inside the chromophore of mEosEM breaks and recombine, causing its excitation wavelength peak and emission wavelength peak to red-shift to 571nm and 581nm, respectively, thereby converting to red fluorescence. In addition, after activation, the photobleaching tolerance of the red light emission of mEosEM is significantly enhanced, which can support long-term dynamic tracking of more than 10 minutes. The red fluorescence state of mEosEM has a balanced quantum yield (0.55) and extinction coefficient (37,000 M -1 cm -1 ), which ensures a high signal-to-noise ratio when 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 a corresponding three-fragment fluorescence complementation system. Summary of the Invention

[0006] In view of this, the primary purpose of the present application is to provide a three-fragment fluorescence complementation system based on the fluorescent protein mEosEM, which is used as a template and is split into three non-luminescent protein fragments AmEosEM, BmEosEM and CmEosEM. These three protein fragments themselves do not have the properties of full-length proteins. When these three fluorescent protein fragments are respectively connected to the first, second, and third test proteins, if the first, second, and third test proteins can interact with each other to spontaneously form trimers or complexes, the three fluorescent protein fragments can fuse and express with each other, so that the three protein fragments form a complete structure and emit fluorescent signals.

[0007] In order to achieve the above objectives, this application adopts the following technical solutions:

[0008] One aspect of the present application provides a three-segment fluorescence complementation system based on the fluorescent protein mEosEM, comprising a first vector, a second vector, and a third vector, wherein the first, second, and third vectors contain nucleotide sequences for expressing first, second, and third target proteins, respectively; the first target protein is a fusion protein of a first test protein and AmEosEM, the second target protein is a fusion protein of a second test protein and BmEosEM, and the third target protein is a fusion protein of a third test protein and CmEosEM;

[0009] The AmEosEM, BmEosEM and CmEosEM are three protein fragments separated from the fluorescent protein mEosEM; the AmEosEM is a protein fragment composed of amino acids from position 1 to position n of the fluorescent protein mEosEM, the BmEosEM is a protein fragment composed of amino acids from position n+1 to position m of the fluorescent protein mEosEM, and the CmEosEM is a protein fragment composed of amino acids from position m+1 to position 227 of the fluorescent protein mEosEM; wherein n=86 and m=151; or, n=151 and m=186; or, n=151 and m=183.

[0010] Another aspect of the present application provides the application of the three-fragment fluorescence complementation system based on the fluorescent protein mEosEM as described above in imaging protein-protein interactions.

[0011] Beneficial effects of this application:

[0012] This application uses the fluorescent protein mEosEM as a template and splits it into three non-luminescent fragments: AmEosEM, BmEosEM, and CmEosEM. The three protein fragments obtained by the splitting method in this application do not have the properties of full-length proteins. When these three fluorescent protein fragments are connected to the first, second, and third test proteins, respectively, if the first, second, and third test proteins can interact with each other to spontaneously form trimers or complexes, the three fluorescent protein fragments can fuse and express with each other, and the three fluorescent protein fragments form a complete structure and emit a fluorescent signal.

[0013] The fluorescence complementary imaging system can be used to verify results through cell transfection at room temperature. It can also be used to verify protein-protein interactions, particularly to specifically verify whether other proteins form trimeric complexes. This system can serve as a simple and efficient system for verifying trimeric interactions. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Schematic diagram of the expression vector used in the examples of this application.

[0015] Figure 2This is a schematic diagram of the structure of the fluorescent protein mEosEM splitting scheme in Examples 2-4 of this application, with blue representing the N-terminus and red representing the C-terminus, wherein: Figure 2 A in the figure is a schematic diagram of the structure of the fluorescent protein mEosEM. Figure 2 B is a schematic diagram of the structure of the splitting scheme in Example 2, Figure 2 C in FIG. 3 is a schematic diagram of the structure of the splitting scheme in Example 3. Figure 2 D in FIG. 4 is a schematic diagram of the structure of the splitting scheme in Example 4.

[0016] 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 in), Example 3 ( Figure 3 C in), 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 G in ).

[0017] Figure 4 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 in), Example 3 ( Figure 4 C in), 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 G in ).

[0018] Figure 5 This is a bar graph of the cell transfection efficiency (i.e., the efficiency of forming fluorescent protein complexes) in Example 5 of the present application.

[0019] 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, where 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, where the first vector: the second vector: the third vector = 1:2:3; Figure 6C in the figure is the gel filtration chromatography result after expression of mEosEM according to the splitting scheme in Example 2 with the first vector: second vector: third vector = 2:3:1.

[0020] 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 SDS-PAGE electrophoresis after expression and purification of mEosEM according to the splitting scheme in Example 4, where the first vector: the second vector: the third vector = 3:2:1; Figure 7 B in the figure is the result of SDS-PAGE electrophoresis after expression and purification of mEosEM according to the splitting scheme in Example 4, where the first vector: the second vector: the third vector = 1:2:3; Figure 7 C in the figure is the result of electrophoresis detection using SDS-PAGE after expression and purification of mEosEM according to the splitting scheme in Example 2, first vector: second vector: third vector = 2:3:1. DETAILED DESCRIPTION

[0021] The following will clearly and completely describe the embodiments of the present application. The technical solutions in the embodiments described below are exemplary and are only possible technical implementations of the present application, not all possible implementations. Those skilled in the art can fully combine the embodiments of the present application to obtain other embodiments without creative work, and these embodiments are also within the scope of protection of the present application.

[0022] The first aspect of the present application discloses a three-fragment fluorescence complementation system based on the fluorescent protein mEosEM, wherein the three-fragment fluorescence complementation system includes a first vector, a second vector and a third vector, wherein the first, second and third vectors contain nucleotide sequences for expressing the first, second and third target proteins, respectively; the first target protein is a fusion protein of the first test protein and AmEosEM, the second target protein is a fusion protein of the second test protein and BmEosEM, and the third target protein is a fusion protein of the third test protein and CmEosEM; the AmEosEM, BmEosEM and CmEosEM are three protein fragments split from the fluorescent protein mEosEM, respectively.

[0023] If the first, second, and third test proteins can interact with each other, they can spontaneously form trimers or complexes within the cell, causing the three fluorescent protein fragments of AmEosEM, BmEosEM, and CmEosEM, which do not emit light on their own, to fuse close to each other, reorganize into a complete structure, and emit a fluorescent signal. Therefore, this three-fragment fluorescence complementation system can be used to verify the interaction between known or unknown proteins, and is particularly suitable for the specific verification of trimeric protein interactions.

[0024] 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:

[0025] MVSAIKPDMRIKLRMEGNVNGHHFVIDGEGTGKPYEGKQTMDLEVKEGGPLPFAFDILTTAFHYGNRVFVKYPDNIQDYFKQSFPKGYSWERSMTFEDGGICNARNDITMEGDTF YNKVRFYGTNFPANGPVMQKKTLKWEPSTEKMYVRDGVLTGDIEMALLLEGGAHYRCDFRTTYKAKEKGVKLPGAHFVDHAIEILSHDKDYNKVKLYEHAVAHSGLPDNARR (SEQ ID NO.1).

[0026] The full-length nucleotide sequence of the fluorescent protein mEosEM in this application is as follows:

[0027] (SEQ ID NO.2).

[0028] In this application, the fluorescent protein mEosEM is split into three non-luminescent protein fragments: AmEosEM, BmEosEM, and CmEosEM. When these three protein fragments are fused with three interacting proteins and co-expressed in the same cell, the three non-fluorescent protein fragments can be pulled together by the interacting proteins, restoring the complete conformation of the fluorescent protein mEosEM, thereby stimulating the generation of a fluorescent signal. Therefore, this three-fragment fluorescence complementation system can be used to study multi-protein interactions through fluorescence imaging reactions under physiological cell temperature conditions.

[0029] In the present application, the first test protein, the second test protein and the third test protein are not particularly limited and can be any protein in the art that interacts and can form a trimer or complex, or any protein that does not interact. The first test protein, the second test protein and the third test protein are respectively connected and fused with the split fluorescent protein fragments to construct corresponding vectors. When designing primers, it is necessary to add a linker sequence or a linker sequence between the first / second / third test protein and the split fluorescent protein fragments as needed. The corresponding design can be performed according to actual needs. Those skilled in the art have such capabilities, so there are no special limitations. When these fusion proteins are co-expressed in the same cell, if the first test protein, the second test protein and the third test protein interact with each other, 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 test protein, the second test protein and the third test protein do not interact, the split fluorescent protein fragments cannot be brought close to each other, and the conformation of the complete fluorescent protein cannot be restored, and no fluorescence will be emitted. Therefore, the three-fragment fluorescence complementation system described in this application can be used to study whether there is interaction between known proteins or unknown proteins.

[0030] In some examples, the first test protein, the second test protein, and the third test protein are trimeric proteins, for example, they can be heterotrimeric monomers or homotrimeric proteins.

[0031] In some preferred examples, the trimeric protein is a homotrimeric protein, and the homotrimeric protein is Foldon.

[0032] Those skilled in the art know that the trimeric protein Foldon is a natural trimeric domain derived from T4 phage fiber protein, composed 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:

[0033] GYIPEAPRDGQAYVRKDGEWVLLSTFLGRS (SEQ ID NO. 3).

[0034] In this application, the full-length nucleotide sequence of the trimeric protein Foldon is as follows:

[0035] ATGGCAGGCTACATTCCTGAAGCCCCTAGAGACGGACAGGCCTATGTCAGAAAGGACGGCGAGTGGGTGCTGCTGAGCACCTTCCTGGGCAGAAGCGGCAGAAGC (SEQ ID NO. 4).

[0036] In a preferred example of the present application, taking the trimeric protein Foldon as an example, Foldon is fused with the three protein fragments split from 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.

[0037] In the present application, the AmEosEM is a protein fragment composed of amino acids 1 to n from the fluorescent protein mEosEM, the BmEosEM is a protein fragment composed of amino acids n+1 to m from the fluorescent protein mEosEM, and the CmEosEM is a protein fragment composed of amino acids m+1 to 227 from the fluorescent protein mEosEM; wherein n=86 and m=151; or, n=151 and m=186; or, n=151 and m=183.

[0038] 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.

[0039] 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; and the third vector contains the nucleotide sequence SEQID NO.7 for expressing the protein fragment CmEosEM.

[0040] In 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; and the third vector contains the nucleotide sequence SEQ ID NO.10 for expressing the protein fragment CmEosEM.

[0041] In 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; and the third vector contains the nucleotide sequence SEQ ID NO.12 for expressing the protein fragment CmEosEM.

[0042] Furthermore, it is understood that the first, second, and third vectors in the three-segment fluorescence complementation system are obtained by inserting nucleotide sequences for expressing the first, second, and third target proteins into 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 test proteins with the nucleotide sequences for expressing the protein fragments AmEosEM / BmEosEM / CmEosEM via a linker sequence. The linker sequence can be specifically designed based on the type of the first / second / third test protein and is not specifically limited. In some examples of the present application, the first / second / third test proteins are all Foldon, and the linker sequence used is GGSGG, whose nucleotide sequence is GGCGGATCCGGAGGC (SEQ ID NO. 13), but is not limited thereto.

[0043] It is understood that, in the present application, the term "operably linked (connected)" or "operably linked (connected)" refers to the functional spatial arrangement of two or more nucleic acid regions or nucleic acid sequences.

[0044] In this application, the expression vector is a genetically engineered vector designed for efficient expression of exogenous genes in host cells and production of corresponding proteins. It is a cloning vector supplemented with expression regulatory elements (such as promoters, ribosome binding sites, and terminators) to enable transcription and translation of the target gene. In this application, the expression vector can be either a prokaryotic or a eukaryotic expression vector. Prokaryotic expression vectors are vectors suitable for bacteria (such as Escherichia coli) that possess a prokaryotic promoter (such as T7 or lac) and an SD sequence. Specific examples include the pET series. Eukaryotic expression vectors are vectors suitable for yeast, mammalian, or insect cells that contain a eukaryotic promoter (such as CMV) and a secretion signal peptide. In this application, mammalian cell expression vectors are preferred. Specific examples include pcDNA3.1. In some specific examples of this application, the expression vector used is PM0147-pIHM_Fc_HRV3C_mEOS, which was constructed in the applicant's laboratory (see Chinese Patent Application Publication No. CN119799784A).

[0045] In this application, the method for constructing the three-segment fluorescence complementation system is not particularly limited and can be implemented using conventional methods in the art. In some examples of this application, the nucleotide sequence of the Foldon-mEosEM fragment (SEQ ID NO. 14) is first obtained by polymerase chain reaction (PCR); the obtained Foldon-mEosEM gene fragment is then inserted into the multiple cloning site of the PM0147-pIHM_Fc_HRV3C_mEOS vector to construct the Foldon-mEosEM vector.

[0046] In addition, it is understood that during the specific design, some specific protein tags can be operably connected as needed, and specific examples include but are not limited to purification tags, etc. For example, in some examples, a his tag (GGCAGCGGACACCACCACCACCACCAC) is added.

[0047] Furthermore, by designing corresponding primers, nucleotide sequences for expressing the first target protein, the second target protein and the third target protein were obtained by PCR amplification from the vector Foldon-mEosEM, and then inserted into the multiple cloning site of the Foldon-mEosEM vector, respectively, to construct the first vector, the second vector and the third vector.

[0048] Among them, those skilled in the art can design primers and select multiple cloning sites according to the inserted protein fragments. Those skilled in the art have such capabilities and will not be elaborated on in detail here.

[0049] The second aspect of the present application discloses the application of the three-fragment fluorescence complementation system based on the fluorescent protein mEosEM as described above in imaging protein-protein interactions.

[0050] In specific applications, the first, second, and third vectors constructed above are co-transfected into cells to be tested in a specific ratio. After a period of incubation, fluorescence imaging is performed. If fluorescence is observed, it indicates that the first, second, or third test proteins interact with each other and form a trimer or complex. If no fluorescence is observed, it indicates that the first, second, or third test proteins do not interact with each other. The ratio of the first, second, and third vectors is not particularly limited and can be determined experimentally by those skilled in the art. For example, in some examples, the mass ratio of the first, second, and third vectors is preferably (1-5):(1-5):(1-5).

[0051] The following are specific embodiments of the present application. It should be noted that the following specific embodiments are only for illustrative purposes and do not limit the scope of the present application in any way.

[0052] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0053] In addition, unless otherwise specified, methods without specific conditions or steps are conventional methods, and the reagents and materials used are all commercially available.

[0054] 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.

[0055] Example 1 Construction of Foldon-mEosEM vector

[0056] 1. PM0147-pIHM_Fc_HRV3C_mEOS expression vector

[0057] The PM0147-pIHM_Fc_HRV3C_mEOS expression vector (full length 6731 bp) used in this example was constructed by our laboratory. For details, please refer to the Chinese patent application with publication number CN119799784A. Figure 1 .

[0058] 2. Digest the PM0147-pIHM_Fc_HRV3C_mEOS expression vector with restriction endonucleases BamH I and Bgl II, and insert the nucleotide sequence of the Foldon-mEosEM fragment into it to obtain the vector. The specific steps are as follows:

[0059] (1) The gene sequence of the Foldon-mEosEM fragment (SEQ ID NO. 14) was obtained by PCR amplification. The primer information involved is shown in Table 1:

[0060] Table 1 Primers and their sequence information

[0061]

[0062] (2) The Foldon-mEosEM gene fragment obtained in step (1) was inserted into the multiple cloning site of the PM0147-pIHM_Fc_HRV3C_mEOS vector using the double enzyme cutting sites BamH I and Bgl II to construct the plasmid Foldon-mEosEM.

[0063] Example 2 Construction of a three-segment fluorescence complementation system (n=86 and m=151)

[0064] In this example, a three-segment fluorescence complementation system based on the fluorescent protein mEosEM was constructed, comprising a first vector, a second vector, and a third vector. The mEosEM split sites were located at positions 86 / 87 and 151 / 152 of mEosEM, resulting in a protein fragment AmEosEM consisting of amino acids 1 to 86 of the fluorescent protein mEosEM, a protein fragment BmEosEM consisting of amino acids 87 to 151 of the fluorescent protein mEosEM, and a protein fragment CmEosEM consisting of amino acids 152 to 227 of the fluorescent protein mEosEM. The specific steps are as follows:

[0065] (1) Construction of the first vector:

[0066] The nucleotide sequence for expressing the first target protein, Foldon-linker-AmEosEM, was obtained by PCR amplification on the plasmid Foldon-mEosEM constructed in Example 1. The nucleotide sequence for expressing Foldon is shown in SEQ ID NO. 4, the nucleotide sequence for linker is shown in SEQ ID NO. 13, and the nucleotide sequence for expressing the protein fragment AmEosEM is shown in SEQ ID NO. 5. The primer information involved is shown in Table 2:

[0067] Table 2 Primers and their sequence information

[0068]

[0069] The nucleotide sequence for expressing the first target protein was inserted into the multiple cloning site of the plasmid Foldon-mEosEM using the double enzyme cutting sites BamH I and Bgl II to construct a first vector.

[0070] (2) Construction of the second vector:

[0071] 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 expressed protein fragment BmEosEM is shown in SEQ ID NO. 6, and the primer information involved is shown in Table 3:

[0072] Table 3 Primers and their sequence information

[0073]

[0074] The nucleotide sequence for expressing the second target protein was inserted into the multiple cloning site of the plasmid Foldon-mEosEM using the double enzyme cutting sites BamH I and Bgl II to construct a second vector.

[0075] (3) Construction of the third vector:

[0076] The nucleotide sequence for expressing the third target protein, namely Foldon-linker-CmEosEM, was obtained by PCR amplification on the plasmid Foldon-mEosEM constructed in Example 1. 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:

[0077] Table 4 Primers and their sequence information

[0078]

[0079] The nucleotide sequence for expressing the third target protein was inserted into the multiple cloning site of the plasmid Foldon-mEosEM using the double enzyme cutting sites BamH I and Bgl II to construct a third vector.

[0080] Example 3 Construction of a three-fragment fluorescence complementation system (n=151 and m=186)

[0081] In this example, another three-segment fluorescence complementation system based on the fluorescent protein mEosEM was constructed, comprising a first vector, a second vector, and a third vector. The mEosEM split sites were located at positions 151 / 152 and 186 / 187 of mEosEM, resulting in a protein fragment AmEosEM consisting of amino acids 1 to 151 of the fluorescent protein mEosEM, a protein fragment BmEosEM consisting of amino acids 152 to 186 of the fluorescent protein mEosEM, and a protein fragment CmEosEM consisting of amino acids 187 to 227 of the fluorescent protein mEosEM. The specific steps are as follows:

[0082] (1) Construction of the first vector:

[0083] 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. The nucleotide sequence of the expressed protein fragment AmEosEM is shown in SEQ ID NO. 8, and the primer information involved is shown in Table 5:

[0084] Table 5 Primers and their sequence information

[0085]

[0086] The nucleotide sequence for expressing the first target protein was inserted into the multiple cloning site of the plasmid Foldon-mEosEM using the double enzyme cutting sites BamH I and Bgl II to construct a first vector.

[0087] (2) Construction of the second vector:

[0088] 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:

[0089] Table 6 Primers and their sequence information

[0090]

[0091] The nucleotide sequence for expressing the second target protein was inserted into the multiple cloning site of the plasmid Foldon-mEosEM using the double enzyme cutting sites BamH I and Bgl II to construct a second vector.

[0092] (3) Construction of the third vector:

[0093] The nucleotide sequence for expressing the third target protein, namely Foldon-linker-CmEosEM, was obtained by PCR amplification on the plasmid Foldon-mEosEM constructed in Example 1. 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:

[0094] Table 7 Primers and their sequence information

[0095]

[0096] The nucleotide sequence for expressing the third target protein was inserted into the multiple cloning site of the plasmid Foldon-mEosEM using the double enzyme cutting sites BamH I and Bgl II to construct a third vector.

[0097] Example 4 Construction of a three-fragment fluorescence complementation system (n=151 and m=183)

[0098] In this example, another three-segment fluorescence complementation system based on the fluorescent protein mEosEM was constructed, comprising a first vector, a second vector, and a third vector. The mEosEM split sites were located at positions 151 / 152 and 183 / 184 of mEosEM, resulting in a protein fragment AmEosEM consisting of amino acids 1 to 151 of the fluorescent protein mEosEM, a protein fragment BmEosEM consisting of amino acids 152 to 183 of the fluorescent protein mEosEM, and a protein fragment CmEosEM consisting of amino acids 184 to 227 of the fluorescent protein mEosEM. The specific steps are as follows:

[0099] (1) Construction of the first vector: refer to the construction of the first vector in Example 3.

[0100] (2) Construction of the second vector:

[0101] 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 expressed protein fragment BmEosEM is shown in SEQ ID NO. 11, and the primer information involved is shown in Table 8:

[0102] Table 8 Primers and their sequence information

[0103]

[0104] The nucleotide sequence for expressing the second target protein was inserted into the multiple cloning site of the plasmid Foldon-mEosEM using the double enzyme cutting sites BamH I and Bgl II to construct a second vector.

[0105] (3) Construction of the third vector:

[0106] The nucleotide sequence for expressing the third target protein, namely Foldon-linker-CmEosEM, was obtained by PCR amplification on the plasmid Foldon-mEosEM constructed in Example 1. 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:

[0107] Table 9 Primers and their sequence information

[0108]

[0109] The nucleotide sequence for expressing the third target protein was inserted into the multiple cloning site of the plasmid Foldon-mEosEM using the double enzyme cutting sites BamH I and Bgl II to construct a third vector.

[0110] Comparative Example 1

[0111] In this comparative example, the splitting site of the fluorescent protein Venus disclosed in the Chinese patent application with publication number CN109385450A was split in mEosEM. The splitting sites are located at positions 149 / 150 and 165 / 166 of mEosEM, and the corresponding vectors were prepared according to the method in Example 3.

[0112] Among them, the primer information involved in the vector construction process is shown in Table 10:

[0113] Table 10 Primers and their sequence information

[0114]

[0115] Comparative Example 2

[0116] In this comparative example, the GFP splitting site disclosed in the Chinese patent application with publication number CN109735569A was split in mEosEM. The splitting sites were located at positions 185 / 186 and 204 / 205 of mEosEM. The corresponding vectors were prepared according to the method in Example 3.

[0117] Among them, the primer information involved in the vector construction process is shown in Table 11:

[0118] Table 11 Primers and their sequence information

[0119]

[0120] Example 5 Identification of Recombinant Green Fluorescent Protein

[0121] 1. 293T cell culture

[0122] 293T cells (CRL-11268 ™ , ACTT) were cultured in complete DMEM (C11885500BT, Gibico) containing 10% fetal bovine serum, 1% GlutaMax, nonessential amino acids, sodium pyruvate, ampicillin, and streptomycin in a 37°C constant-temperature cell culture incubator with 5% CO2. The culture medium was replaced and subcultured according to the cell growth status.

[0123] The source of information on components in the culture medium is shown in Table 12:

[0124] Table 12 Culture medium composition information

[0125]

[0126] 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 culture incubator for 1 min until the cells are completely detached; add 9 mL of DMEM culture medium, pipet and mix the cells, transfer 1 mL to a new culture dish, and add 9 mL of fresh culture medium to continue culturing.

[0127] 2. 293T cell transfection

[0128] One day before the transfection experiment, 5 × 10 6 293T cells were suspended in 10 mL of complete culture medium and cultured overnight at 37°C in a cell culture incubator with 5% CO2 to allow the cells to reach a confluency of 70%-80%. Before transfection, the cells were digested normally and 10 mL of complete culture medium was added to disperse the digested cells, suspend and disperse the cells, and count the cells. 2.5 mL was added to each well of a 6-well plate, and 0.67×10 6 The number of cells was plated.

[0129] 0.3 μg each of the first, second, and third vectors from Example 2 were added to a centrifuge tube containing 100 μl of Opti-MEM medium. A transfection liposome reagent was then added (the mass-to-volume ratio of plasmid to transfection liposome reagent was 1:1). The incubated plasmid-transfection reagent mixture was evenly dispersed and added to 293T cells. The cells were cultured in a cell culture incubator at 37°C and 5% CO₂ for 48 h.

[0130] The first vector, the second vector and the third vector in Examples 3 and 4, and Comparative Examples 1 and 2 were all transfected using the same transfection method as above.

[0131] A negative control (blank) and a positive control (plasmid Foldon-mEosEM transfection) were set up at the same time. The plasmid Foldon-mEosEM transfection used the same transfection method as above.

[0132] 3. Sample preparation for flow cytometry

[0133] (1) Prepare FACS solution: add 1 mL of FBS solution to 50 mL of PBS buffer.

[0134] (2) Remove the cells from the incubator, aspirate and discard the culture medium above the cells, add 1 mL of FACS solution to each well of the plasmid transfection well and 1 mL of FACS solution to the control well.

[0135] (3) Use a pipette to disperse the cells and dispense the cells into flow cytometry tubes in each well, with each tube containing 1 mL. Centrifuge at 2000 rpm and 4°C for 6 min.

[0136] (4) After centrifugation, discard the supernatant and add 500 μl of FACS solution to each tube to resuspend the cells; centrifuge at 2000 rpm and 4°C for 6 minutes.

[0137] (5) Prepare 2% fixative during centrifugation: add 5 mL of 4% tissue cell fixative to 5 mL of 1× PBS solution and vortex to mix.

[0138] (6) After centrifugation, discard the supernatant, add 500 μl of 2% fixative to each tube, vortex to mix, and wait for the instrument to detect.

[0139] in, Figure 3 and Figure 4 The test results of the three-segment fluorescence complementation system in Examples 2-4 and Comparative Examples 1 and 2 are shown respectively. If a functional fluorescence is formed, a fluorescence signal can be seen in FITC. If the splitting is unsuccessful, no fluorescence signal can be seen. Figure 3 and Figure 4 It can be seen that Examples 2, 3 and 4 and the positive control can see fluorescent signals in FITC, while Comparative Examples 1 and 2 and the negative control cannot see fluorescent signals in FITC, indicating that the splitting scheme in this application is feasible and successful.

[0140] Furthermore, the percentage of FITC fluorescence positive cells in all cells was calculated as follows: Figure 5 As shown in the cell transfection efficiency chart, the transfection efficiencies of Examples 2-4 were 24.2%, 35%, and 33.5%, respectively; while the transfection efficiencies of Comparative Examples 1 and 2 were 0.51% and 0.54%, respectively. This indicates that the split sites used in Comparative Examples 1 and 2 were unable to fully form the mEosEM structure and function.

[0141] Example 6 Protein Purification

[0142] 1. Solution preparation

[0143] (1) Cell lysis buffer preparation (100 mL): 4 ml 500 mM Imidazole Buffer + 8.18 g NaCl + 5 ml 10% V / W NP-40 + 10 ml 5% V / W Sodium deoxycholate + 0.736 g Glu (glutamic acid) + 0.84 g Arg (arginine).

[0144] (2) Imidazole gradient elution buffer: Use 500mM Imidazole Buffer and PBS to prepare low-concentration imidazole elution buffers with concentrations of 20mM and 50mM, respectively, and adjust the pH to 7.6.

[0145] 2. Transfection of 293F cells (FreeStyle™ 293-F cells, Cat. No. R79007, Gibco)

[0146] (1) Cell counting: ordinary bright field / trypan blue staining counting (add 25 μL to the slide).

[0147] (2) Cell dilution: dilute the cells to 1×10 6 / mL (0.8~1×10 6 The transfection concentration was (floating range: cells / mL).

[0148] (3) Plasmid filtration: The plasmid to be transfected (1 mg plasmid corresponds to 1 L of cells (concentration 1×10 6 The plasmids were added to 1 mL of OptiPRO™ SFM (GIBCO, Cat. No. 12309019) and mixed thoroughly. The Optim medium containing the plasmids was sterilized by filtration using a 2.5 mL syringe and a 0.22 μm filter membrane.

[0149] (4) Plasmid packaging: Slowly add the transfection reagent (LiFect293™ Transfection Reagent, LIFESCT Cat.#: M0002-01) (1 μg of plasmid corresponds to 1 μL of transfection reagent) into the filtered Optim medium containing the plasmid, invert to mix, and let it stand at room temperature for 5 minutes.

[0150] (5) Transfect cells: Add all the incubated plasmids to the diluted cells (1×10 6 cells / mL).

[0151] (6) Harvesting cell supernatant: 6-7 days after successful transfection, collect the cell supernatant by centrifugation at 12000 rpm for 10 min, and filter through 0.22 μm for protein purification.

[0152] 3. Primary purification of protein: nickel column purification

[0153] (1) Collect the 293F cells transfected with the three plasmids of Foldon-mEOS (i.e., the first vector, the second vector, and the third vector), centrifuge at low temperature and high speed for 15 minutes, and discard the supernatant to obtain the precipitate sample.

[0154] (2) Resuspend the pellet in 10 volumes of cell lysis buffer and lyse at 4°C for 60 min.

[0155] (3) Centrifuge the lysed cells at low temperature and high speed, aspirate the supernatant and filter.

[0156] (4) Add pretreated nickel column gel to the supernatant filtrate and incubate at 4°C on a shaker at 130 rpm for 1 h.

[0157] (5) Transfer the combined liquid to a gravity column, wait for the liquid to drain, and collect the flow-through.

[0158] (6) Elution with imidazole gradient eluent: ① Take 30 mL of 20 mM IMD with PBS, pH 7.6 eluent for elution; ② After the liquid has drained, take 30 mL of 50 mM IMD with PBS, pH 7.6 eluent for elution; ③ After the liquid has drained, take 30 mL of 500 mM IMD with PBS, pH 7.6 eluent for elution again.

[0159] (7) Collect the flow-through and the elutions with different concentrations of imidazole from step (6), transfer the elutions to concentration tubes, and perform low-temperature centrifugal ultrafiltration at 3500 rpm.

[0160] (8) After the eluate is concentrated to a volume of 1 mL, observe whether there is fluorescence in the solution. If a solution with fluorescence is observed, proceed to the next step of purification.

[0161] 4. Protein purification: gel filtration chromatography purification.

[0162] (1) Sterilization: The protein solution concentrated to a volume of 1 mL was filtered through a 0.22 μm filter membrane and centrifuged at 14,000 rpm for 5 min.

[0163] (3) Equilibration system: Use PBS to equilibrate the SEC system, chromatography column and sample loop in advance.

[0164] (4) Inject the sample into the sample loop of the SEC system and start the purification process.

[0165] (5) Sample the separated and purified protein according to the SEC results, perform SDS-PAGE electrophoresis detection, and concentrate the target sample and detect the concentration.

[0166] 5. SDS-PAGE electrophoresis detection

[0167] (1) Calculate the molecular weight of the target protein and predict the peak range of the target protein.

[0168] (2) Based on the SEC chart, concentrate the protein solution in the target interval.

[0169] (3) Sampling: Mix the protein concentrate with 5× loading buffer at a volume ratio of 4:1.

[0170] (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.

[0171] (5) After the sample is centrifuged at low speed to mix, add the protein gel well, add electrophoresis solution to the inside and outside of the electrophoresis tank, and perform electrophoresis at a constant voltage of 140V for 60 minutes.

[0172] (6) After stripping the electrophoresis gel, place it in Fast Blue protein rapid staining solution and stain it evenly for 3 hours.

[0173] (7) Place the stained protein gel in clean water and decolorize for 60 minutes.

[0174] 6. Protein yield calculation

[0175] (1) Transfer the target protein solution that has been purified and concentrated in two steps into an EP tube and vortex mix.

[0176] (2) Use an ultra-micro UV spectrophotometer, click the A280 mode, enter the theoretical extinction coefficient of the protein, and perform the test.

[0177] (3) Blank: Take 1 μL of PBS and drop it into the detector for blanking.

[0178] (4) Measurement: After the blank is completed, wipe off the blank solution, take 1 μL of protein solution and drop it into the detector, click on the measurement, and record the concentration of the detected protein solution.

[0179] (5) Use a pipette to aspirate the test protein solution volume multiple times and calculate the final protein yield based on the volume and concentration of the protein solution.

[0180] Figure 6 The experimental results of gel filtration chromatography in this embodiment are shown. Figure 6 A in the figure is the gel filtration chromatography result after expression of mEosEM transfected using the splitting scheme in Example 4 (according to the mass ratio of the first vector, the second vector and the third vector being 3:2:1). Figure 6 B in the figure is the gel filtration chromatography result after expression of mEosEM transfected using 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 the figure is the gel filtration chromatography result after expression of mEosEM transfected using 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).

[0181] It can be seen that after adopting the splitting scheme of the present application, the three-split fluorescent protein can be purified and can be purified with other proteins through molecular sieve chromatography gel, and further separation and purification can be achieved subsequently.

[0182] Figure 7 The results of SDS-PAGE electrophoresis in this embodiment are shown. Figure 7 A in the figure is the result of electrophoresis detection using SDS-PAGE after expression and purification of mEosEM transfected using the splitting scheme in Example 4 (according to the mass ratio of the first vector, the second vector and the third vector being 3:2:1). Figure 7 B in the figure is the result of electrophoresis detection using SDS-PAGE after expression and purification of mEosEM transfected using 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 the figure is the result of electrophoresis detection using SDS-PAGE after expression and purification of mEosEM transfected using 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).

[0183] It can be seen that after separation and purification, the band size and position are in line with expectations.

[0184] The above examples demonstrate that the splitting schemes of this application can be successfully expressed in vitro through protein interactions and can be purified through expression, indicating that the three splitting schemes for the fluorescent protein mEosEM in this application are successful and feasible. This is of great significance for studying protein interactions, especially for specifically verifying whether other proteins are trimeric complexes. This system can be used as a simple, efficient system for verifying trimeric interactions.

[0185] It should be noted that the present application is not limited to the above-mentioned embodiments. The above-mentioned embodiments are merely examples, and any embodiments having substantially the same structure and effect as the technical concept within the scope of the present application are all included in the technical scope of the present application. In addition, without departing from the scope of the present application, any other embodiments that can be conceived by those skilled in the art and that combine some of the constituent elements in the embodiments are also included in the scope of the present application.

Claims

1. A three-segment fluorescence complementation system based on the fluorescent protein mEosEM, characterized in that: It includes a first vector, a second vector and a third vector, wherein the first, second and third vectors contain nucleotide sequences for expressing the first, second and third target proteins, respectively; the first target protein is a fusion protein of the first test protein and AmEosEM, the second target protein is a fusion protein of the second test protein and BmEosEM, and the third target protein is a fusion protein of the third test protein and CmEosEM; The AmEosEM, BmEosEM and CmEosEM are three protein fragments respectively 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 from position 1 to position n of the fluorescent protein mEosEM, the BmEosEM is a protein fragment composed of amino acids from position n+1 to position m of the fluorescent protein mEosEM, and the CmEosEM is a protein fragment composed of amino acids from position m+1 to position 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-segment 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-segment 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-segment 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-segment 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 according to claim 8, characterized in that: The expression vector is a prokaryotic expression vector or a mammalian cell expression vector.

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

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