WO2012067402A9 - Red fluorescent protein fragment having a self-assembly activity, method for preparing same, and method for analyzing protein-protein interactions using same - Google Patents

Abstract

The present invention relates to a red fluorescent protein fragment having a self-assembly activity, to a method for preparing same, and to a method for analyzing protein-protein interactions using same. The red fluorescent protein fragment (mPlum 1 E16V) according to the present invention is prepared by coupling the β-strand at position 1 of the red fluorescent protein (mPlum) after the β-strands at positions 2 to 11 via a flexible peptide linker so as to prepare a circularly permutated mPlum protein (CpmPlum), replacing glutamic acid (Glu) at position 16 of the amino acid sequence of the β-strand at position 1 of the circularly permutated mPlum protein (CpmPlum) with valine (Val) so as to prepare a mutant (CpmPlum E16V), separating the mPlum 1 peptide (mPlum 1 E16V), which is mutated alone, from mPlum 2-11 protein and self-assembling the separated mPlum 1 peptide and mPlum 2-11 protein in order to emit a red fluorescent signal, thus enabling the red fluorescent protein fragment to have a self-assembly activity. Accordingly, the red fluorescent protein fragment (mPlum 1 E16V) having a self-assembly activity according to the present invention may serve as a biosensor that can be effectively used in analyzing the position of a protein in a living cell and the function of a protein such as protein-protein interaction.

Description

Red fluorescent protein fragment having self-binding activity, preparation method thereof and analysis method of protein interaction using same

The present invention relates to a red fluorescent protein fragment having self-binding activity, a method for preparing the same, and a method for analyzing a protein using the same.

Proteins are macromolecules that make up the body of every living organism and are one of the most basic cellular components as a link of many amino acids. Since protein plays a role in mediating various reactions and phenomena in vivo, it is very important to analyze and confirm the function of the protein. In addition, since proteins are expressed or regulated mainly through interaction with other proteins, identifying protein interactions is a research field that must be preceded in order to properly understand various life phenomena occurring in vivo. In particular, analysis of protein interactions in living cells has a biological significance because it can reflect the actual environment as much as possible.

As such, as the importance of understanding protein interactions increases, experimental techniques for analyzing them have also been developed. In general, experiments performed to examine protein interactions include immunoprecipitation and protein chips.

In vivo, proteins perform their functions by binding to other proteins, so when one protein is purified, other proteins that bind to it are also purified. In other words, if Y is the other protein that binds to X, a compound precipitate of X / Y / X-antibody can be obtained when the antibody against X is added. When X is detected after electrophoresis analysis of the precipitate, Can be analyzed biochemically. Nowadays, a lot of methods for analyzing protein interactions using mass spectrometry on unknown proteins purified through immunoprecipitation are used. In recent years, rapid advances in mass spectrometry have made it possible to identify proteins with only a small amount of protein (up to 100 fmol or less). Recently, protein interaction analysis has been performed at the individual level.

After the protein chip is immobilized on the slide glass, the protein interaction can be analyzed through optical shape analysis, mass spectrometry, fluorescence analysis, and electrochemical analysis. In particular, since binding affinity between proteins can be measured under various conditions such as ionic strength and pH, it is recognized as an essential technique for understanding interaction mechanisms such as changes in interactions according to various parameter changes.

However, all of the above-described methods for analyzing protein interactions are different from actual in vivo interactions because they are conducted under in vitro conditions, and in the case of immunoprecipitation, there is a high possibility of loss in the purification of proteins. It is difficult to detect without protein interactions with very strong binding, and protein chips have many false positive results despite the advantage of easy analysis of various protein interactions at once.

In addition, the yeast two-hybrid system, a method that has been widely used recently, is a method that can analyze protein interactions through transcriptional activity of receptor genes in living yeast cells rather than extracellularly. And genome fusion libraries have been widely used as a way to explore protein interactions. The yeast two-hybrid system has the advantage of being able to see protein interactions in vivo, contributing greatly to the study of protein interactions in cells of higher animals. However, the yeast two-hybrid system is useful for analyzing protein interactions inside the nucleus because it is a system of complementary binding of transcription factors, but the disadvantage of showing positive error results when analyzing protein interactions outside the nucleus. have.

Thus, a new concept of protein interaction analysis system was developed using the principle of fluorescence resonance energy transfer (FRET). Fluorescent proteins absorb and excite light in different wavelength ranges, and once their energy is released as light or heat, they return to the ground state and emit a unique wavelength range for each fluorescent protein. The fluorescence resonance energy transfer principle is that when two different fluorescent proteins are within 10-100 kHz, the light emitted after the short wavelength fluorescent protein is excited induces the excitation of the long wavelength fluorescent protein to emit fluorescence. That is, by attaching two different fluorescent proteins to each other after the two proteins to be examined for interaction, and detecting fluorescence due to the fluorescence resonance energy transfer phenomenon that occurs when the two proteins are within 10 to 100 에 by protein interaction. It is possible to analyze protein interactions. This system has the advantage of being able to analyze the dynamic interaction of proteins in living cells, but only if the distance between the two fluorescent proteins is very close and detectable, and if overexpression of the protein is required to detect subtle changes in fluorescence wavelengths. There is a disadvantage that expensive equipment is required for fluorescence resonance energy transfer analysis.

Therefore, recently, biomolecular protein fluorescence complementation, which has improved the limitations of the aforementioned conventional protein assays, has attracted attention. Two-molecule fluorescence complementation analysis is a technique that uses the phenomenon of fluorescence by dividing a fluorescent protein into two fragments, but when the two fragments are far apart, they do not fluoresce, but when they come close to each other, they bind together to form an intact fluorescent protein complex. In this study, fusion proteins with fluorescent protein fragments attached to two proteins to study protein interaction are expressed in a cell, and when the two proteins interact with each other, the fluorescent signals linked to each other are measured. That's how. Since these two-molecule fluorescence complementary assays have been successfully used to analyze the interactions and intracellular location of bZIP and Rel family proteins, they have been applied to various individuals such as animals, plants, and yeasts. In addition, it is possible to measure the fluorescence signal through a relatively simple equipment, and because the signal intensity in the cell is strong, it is possible to observe where the protein interaction occurs in the cell has many advantages over the conventional method have.

In addition, the two-molecule fluorescence complementation assay may use various receptor proteins depending on the purpose of the study. This is a useful technique for analyzing protein-protein interactions, where the choice of fluorescent proteins that can separate into two fragments is important.

Isolate protein-based receptor systems have been widely used to monitor various protein-protein interactions in vitro and in vivo . Receptors based on two-molecule fluorescence complementation assays known to date include dihydrofolate reductase (DHFR), β-galactosidase, β-lactamase, TEV protease, luciferase, green fluorescent proteins (GFPs), and modified proteins thereof. . These receptor proteins are separated into two fragments that do not spontaneously bind. In other words, the isolated fragments are bound only when the receptor proteins fused together to generate a signal that the protein partner can interact to detect.

Recently, fluorescent proteins with several self-cleaving activities have been used for two-molecule fluorescence complementation assays. Representative self-cleaving fluorescent proteins include self-associating fragments of GFP, which have been developed from very stable "superfolder" GFPs. That is, the last β-strand (GFP 11, amino acids 214-230) of the GFP protein spontaneously binds to large GFP fragments (GFP 1-10, amino acids 1-214) and successfully forms mature chromophores. Such assays include cellular protein solubility testing, synthetic structure of GFPs, and small molecule detection. More importantly, separating and self-binding one β-strand from the 11 β-strand barrel form of fluorescent protein for chromophore formation can provide great potential for protein-peptide tagging and detection.

In addition, YN155 and YC155, fragments cut from yellow fluorescent protein (YFP) at amino acid 155, and VN155, cut at amino acids 155 or 173, which is a variant of yellow fluorescent protein. VC155 or VN173 and VC173 fragments are used. In addition, the red fluorescent protein that emits light having a long wavelength is used fragments 1 to 159 and 160 to 237 mCherry fluorescent protein fragments cut between amino acid sequences 159 and 160 of the mCherry fluorescent protein.

As mentioned above, two-molecule fluorescence complementation assays are powerful assays capable of analyzing the function of proteins in living cells at low cost. However, the two-molecule fluorescence complementation assay has technically complementary matters. In two-molecule fluorescence complementation assays, there is room for false positive reactions due to the fusion of only receptor fragments rather than interactions between target proteins. In contrast, detection of the actual target protein interaction, depending on the topology of the receptor fragment or the nature of the target protein, or the nature of the flexible linker linking the target protein to the receptor fragment It is also possible that false negatives do not occur. Therefore, there is a need for the development of fluorescent proteins with self-binding activity in a direction that blocks this possibility.

Red fluorescent proteins, on the other hand, are thought to be promising in the field of tissue imaging and cellular biosensors due to emission "optical windows" of 600 nm and above. Of these red fluorescent proteins, mPlum, a monomeric fluorescent protein with one of the most redshifted emission spectra (maximum emission at 649 nm), is excited at 590 nm, and has two major residues (Glu 16 and Ile 65). Is dominated by However, the mPlum having such spectroscopic characteristics is rarely used in two-molecule fluorescence complementation analysis, and there is almost no research on this. Therefore, after confirming that the mPlum fragment, which is a red fluorescent protein, has self-binding activity, applying the mPlum fragment to a bimolecular fluorescence complementation assay is considered to contribute greatly to the study of protein interaction.

While studying the fluorescent protein having self-binding activity, the inventors linked the β-strand 1 of the red fluorescent protein (mPlum) through the flow peptide linker followed by the 11-11 β-strand through a flow peptide linker. After preparing (CpmPlum), mutant (CpmPlum E16V) was prepared by replacing Glu (glutamic acid) of No. 16 of the amino acid sequence of β-strand 1 with Val (valine), and mutated mPlum 1 peptide alone (mPlum 1). E16V) was isolated from the mPlum 2-11 protein to obtain a red fluorescent protein fragment (mPlum 1 E16V), and then the red fluorescent protein fragment (mPlum 1 E16V) self-bound with the mPlum 2-11 protein to emit a red fluorescent signal. It was confirmed to have self-binding activity and the present invention was completed.

The present invention aims to provide a red fluorescent protein fragment having self-binding activity, a method for preparing the same, and a method for analyzing a protein using the same.

The red fluorescent protein fragment (mPlum 1 E16V) according to the present invention is linked to the β-strand 1 of the red fluorescent protein (mPlum) through the flow peptide linker behind the β-strand 2-11, the ordered mPlum protein (CpmPlum) After the preparation, the mutant (CpmPlum E16V) was prepared by replacing 16 Glu (glutamic acid) of Val (valine) in the amino acid sequence of β-strand 1 of the ordered mPlum protein (CpmPlum) with val (valine) MPlum 1 peptide (mPlum 1 E16V) is isolated from mPlum 2-11 protein and self-binds with mPlum 2-11 protein to emit red fluorescent signal, thereby having self-binding activity. Thus, the red fluorescent protein fragment (mPlum 1 E16V) with self-binding activity according to the present invention can be used as a biosensor as a useful tool for analyzing protein function such as protein location in living cells, protein-protein interactions, etc. Can be.

1 is a diagram showing the amino acid sequence of full-length mPlum and permuted mPlum protein (CpmPlum) (mPlum 1 (red), mPlum 2-11 (blue), and Glu16 (yellow)).

FIG. 2 shows fluorescence images of E. coli colonies expressing several mPlum fragments.

FIG. 3 is a diagram showing normalized absorption spectra (solid line) and fluorescence spectra (dashed line, excitation at 570 nm) of mPlum, mPlum E16V, CpmPlum E16V, mPlum fragments.

Figure 4 is a diagram showing the manufacturing process of mPlum fragment having a self-binding activity according to the present invention.

Figure 5 shows the in vitro complementarity of mPlum 2-11 and synthetic mPlum 1 E16V peptide.

Figure 6 shows the in vivo complementarity of several mPlum 1 peptide mutants and mPlum 2-11.

FIG. 7 shows the fluorescence spectra of mPlum 1 E16V C-1 peptides bound to mPlum 2-11 and the fluorescence spectra of CmPlum E16V bound to mPlum 2-11.

8 shows fluorescence images of HeLa cells expressing various mPlum fragments.

9 shows fluorescence images of tetracycline-regulated HeLa cells that sequentially express several mPlum fragments.

10 shows fluorescence images of tetracycline-regulated HeLa cells that sequentially express several mPlum fragments at the time of induction of tetracycline.

FIG. 11 is a diagram illustrating the fiberization of α-syn, α-syn-mPlum 1 E 16 V, and MBP-mPlum 1 E 16 V. FIG.

12 shows fluorescence images of tetracycline-regulated HeLa cells.

The present invention is characterized in that Glu (glutamic acid) of 16 of the amino acid sequence of β-strand 1 of the red fluorescent protein (mPlum) is replaced with Val (valine), and has a self-binding activity having the amino acid sequence of SEQ ID NO: 1. A red fluorescent protein fragment (mPlum 1 E16V) is provided.

The present invention also provides a gene encoding the red fluorescent protein fragment (mPlum 1 E16V).

The present invention also provides an expression vector comprising the gene.

The present invention also provides a host cell comprising the expression vector.

In addition, the present invention provides a protein detection kit comprising the red fluorescent protein fragment (mPlum 1 E16V).

In addition, the present invention

1) preparing the ordered mPlum protein (CpmPlum) by connecting the β-strand 1 of the red fluorescent protein (mPlum) through the flow peptide linker behind the β-strand 2-11;

2) preparing a mutant (CpmPlum E16V) by substituting Val (valine) for 16 Glu (glutamic acid) in the amino acid sequence of β-strand 1 of the ordered mPlum protein (CpmPlum),

3) separating the mutant (CpmPlum E16V) into mPlum 2-11 fragment and mPlum 1 E16V fragment, and

4) It provides a method for producing a red fluorescent protein fragment (mPlum 1 E16V) having a self-binding activity, comprising the step of self-binding the separated mPlum 2-11 fragment and mPlum 1 E16V fragment.

The present invention also expresses mPlum 1 E16V (MBP-mPlum 1 E16V) and His-tagged mPlum 2-11 (His-mPlum 2-11) fused with a maltose-binding protein in a host cell. The present invention provides a method for analyzing protein interaction using a red fluorescent protein fragment having self-binding activity (mPlum 1 E16V), which is characterized by detecting a red fluorescent signal in a host cell.

Hereinafter, the present invention will be described in detail.

The red fluorescent protein fragment (mPlum 1 E16V) having self-binding activity according to the present invention can be obtained by linking the β-strand 1 of the red fluorescent protein (mPlum) through the flow peptide linker behind the β-strand 2-11. After preparing the permuted mPlum protein (CpmPlum), Glu (glutamic acid) of 16 of the amino acid sequence of β-strand 1 of the permuted mPlum protein (CpmPlum) was replaced with Val (valine) and the mutant (CpmPlum E16V). ) And the mutated mPlum 1 peptide (mPlum 1 E16V) alone was isolated from the mPlum 2-11 protein. The amino acid sequence of the isolated red fluorescent protein fragment (mPlum 1 E16V) is represented by SEQ ID NO: 1.

Co-expression of mPlum 1 fragment and mPlum 2-11 fragment in E. coli and the ordered mPlum protein (CpmPlum) do not emit red fluorescence signal, so mPlum 1 fragment and mPlum 2-11 fragment have self-binding activity. It can be seen that it does not have. However, CpmPlum mutants (CpmPlum E16V) sequenced in E. coli and co-expression of mPlum 2-11 and mPlum 1 E16V emit red fluorescent signals. Thus, it can be seen that the mPlum 2-11 fragment is self-linked with the mPlum 1 E16V peptide, and the mPlum 1 E16V peptide has self-binding activity.

In addition, the in vitro complementarity between the mPlum 2-11 protein and the mPlum 1 E16V peptide provided that the higher concentration (4 μM) of the mPlum 1 E16V peptide provided faster complementarity, depending on the change in urea concentration (0.2-0.8 M). Equilibrium is reached after 24 hours. In addition, the mutant (mPlum 1 E16V) replacing Glu 16 of the mPlum 1 peptide with Val (valine) shows high levels of in vivo fluorescence complementarity, whereas Glu 16 of the mPlum 1 peptide is expressed as Ile (isoleucine) or Met. Mutants replaced by (methionine) (mPlum 1 E16I, mPlum 1 E16M) show low levels of in vivo fluorescence complementarity and other mutants do not show fluorescence signals. Thus, it can be seen that the mPlum 1 E16V peptide has good self-binding activity.

In addition, it was confirmed that the minimum residue of mPlum 1 E16V peptide for self-binding with mPlum 2-11 is 18 amino acids, which is the critical point of maximum self-binding. That is, it was confirmed that removing only one residue from the peptide mPlum 1 E16V having 18 amino acid sequences significantly interfered with fluorescence complementation.

In addition, mPlum and CpmPlum E16V showed red fluorescent activity in HeLa cells, whereas mPlum 2-11 fragment alone did not show fluorescent activity. However, the co-transformers of mPlum 2-11 and MBP-mPlum 1 E16V peptides show bright red fluorescence. Thus, it can be seen that the mPlum 1 E16V peptide tag can be used for tagging and detecting proteins in mammalian cells (HeLa cells).

In addition, MBP protein tagged in mPlum 1 E16V peptide that is not regulated by tetracycline is expressed immediately, whereas expression of mPlum 2-11 protein is inhibited by tetracycline regulation. In addition, wild-type mPlum protein shows a red fluorescence signal upon tetracycline induction. Thus, fluorescent labeling of MBP-mPlum 1 E16V peptides was successfully achieved by adding tetracycline to induce mPlum 2-11 protein. In addition, the red fluorescence signal of mPlum is clearly observed within 6 hours after tetracycline induction, and 8 hours are required for mPlum 2-11 to bind to MBP-mPlum 1 E16V peptide and form a mature chromophore.

As described above, the red fluorescent protein fragment (mPlum 1 E16V) according to the present invention is sequenced by connecting the β-strand 1 of the red fluorescent protein (mPlum) through the flow peptide linker behind the β-strand 2-11 times. After preparing the mPlum protein (CpmPlum), the mutant (CpmPlum E16V) was replaced by Val (valine) by replacing Glu (glutamic acid) of No. 16 of the amino acid sequence of β-strand 1 of the circularly ordered mPlum protein (CpmPlum). The mPlum 1 peptide (mPlum 1 E16V), prepared and mutated alone, is isolated from the mPlum 2-11 protein and self-binds with the mPlum 2-11 protein to emit a red fluorescent signal, thereby having self-binding activity. Thus, the red fluorescent protein fragment (mPlum 1 E16V) with self-binding activity according to the present invention can be used as a biosensor as a useful tool for analyzing protein function such as protein location in living cells, protein-protein interactions, etc. Can be.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the examples.

Example 1  Preparation of Red Fluorescent Protein (mPlum)

1. Preparation of the ordered mPlum protein (CpmPlum)

Genes encoding mPlum fragments such as full length mPlum and mPlum 2-11 (residues 25-216) were amplified by PCR from the commercial mPlum gene (Clontech). Then, to express the recombinant protein in Escherichia coli , the PCR product was cloned with pET28a expression vector (Novagen). For bacterial expression of the mPlum 1 peptide (residues 6-23), the mPlum 1 peptide was fused to the C-terminus of the maltose binding protein (MBP) with the flow peptide linker (GGSGGT) and cloned with the pET21a expression vector (Novagen). The ordered mPlum protein (CpmPlum) was then prepared by fusing the mPlum 1 fragment to the C-terminus of the mPlum 2-11 fragment via a flow peptide linker (GGTGGSGGTGGS). The amino acid sequences of the full length mPlum and the ordered mPlum protein (CpmPlum) are shown in SEQ ID NOs: 2 and 3, respectively, and are shown in FIG. 1 [mPlum 1 (red), mPlum 2-11 (blue), and Glu16 (yellow). ].

In order to confirm the self-binding activity of the prepared permutated mPlum protein (CpmPlum), E. coli BL21 (DE3) cells in which the expression plasmid lived were grown at 37 ° C. until OD ₆₀₀ = 0.6. The protein was then induced with 1 mM IPTG (isopropyl-β-D-thiogalactopyranoside) and incubated overnight at 25 ° C. After sonication-based disruption of the induced cells, the proteins were purified by Ni-chelating column. Purified protein was dialyzed in PBS buffer and stored at -20 ° C before use.

In vivo complementarity of mPlum fragments was tested by co-expressing the mPlum fragments in E. coli BL21 (DE3). First, E. coli BL21 (DE3) cells were transformed with expression vectors of mPlum 2-11 (in pET28a, kanamycin resistant) and MBP-fused mPlum 1 peptide (in pET21a, ampicillin resistant) and supplemented with ampicillin and kanamycin Placed on nitrocellulose membrane in LB agar medium. After growing overnight at 37 ° C., the membranes were transferred to LB plates containing 1 mM IPTG to induce proteins and incubated at 22 ° C. for 24 hours. Fluorescence images of E. coli colonies were obtained with a luminescence imaging analyzer LAS 3000 (FujiFilm) using a 575 nm excitation filter and a 670 nm emission filter.

Fluorescence images of E. coli colonies expressing several mPlum fragments are shown in FIG. 2.

As shown in FIG. 2, red fluorescent protein (mPlum) emitted red fluorescent signal in E. coli , but co-expression and permuted mPlum protein (CpmPlum) of mPlum 1 fragment and mPlum 2-11 fragment showed red fluorescence. It did not emit a signal. Thus, it was confirmed that the mPlum 1 fragment and the mPlum 2-11 fragment do not have self-binding activity.

2. Preparation of Mutants of Circularly Ordered mPlum Protein (CpmPlum) [CpmPlum E16V]

A library of randomly mutated permutated mPlum proteins (CpmPlum) was prepared by error-prone PCR (GeneMorph II Random Mutagenesis Kit, STRATAGENE). PCR products were cloned into pBAD-A expression vector. Library plasmids were transformed into TOP10 electroshock E. coli cells and plated directly on nitrocellulose membrane. After growing overnight at 37 ° C., the membranes were transferred to LB plates containing 0.02% L-arabinose and protein induced. Protein expression and fluorophore formation in E. coli colonies on the membrane were monitored at 16 ° C. for several days. Fluorescent colonies were selected from 6 × 10 ⁴ libraries and 13 mutants (CpmPlum E16V) were screened. Amino acid substitutions of the 13 screened mutants (CpmPlum E16V) were analyzed and are shown in Table 1 below.

Table 1

Clone	Amino acid substitutions
One	E230V
2	H148Y, M158V, E194V, H197Y, E230V
3	S88T, E230V
4	G102C, R125W, E218G, E230V, V236M
5	E218N, E219K, E230V
6	R227P, E230V
7	D150N, E230V
8	K229R, E230V
9	E218Q, E230V
10	R227P, E230V
11	S215N, E218K, E230V
12	K97M, E217G, E230V
13	N74T, E230 V

As shown in Table 1, one common mutation (Glu 230 to Val, E230V) corresponding to Glu 16 of mPlum was observed in all 13 screened CpmPlum mutants (CpmPlum E16V).

3. Self-Binding of mPlum 1 E16V Peptide and mPlum 2-11 Protein

The mutated mPlum 1 peptide alone (mPlum 1 E16V) was isolated from the mPlum 2-11 protein. MBP fused mPlum 1 peptide was then co-expressed with His tagged mPlum 2-11 protein and purified using SDS-PAGE. Bound fluorescent protein was purified in duplicate using fused MBP and His tags. Purified mPlum 2-11 protein spontaneously bound with chemically synthesized mPlum 1 peptide (mPlum 1 E16V). Next, the absorption spectra and fluorescence spectra of the purified proteins were measured.

Fluorescence images of E. coli colonies expressing the ordered CpmPlum mutants (CpmPlum E16V) or mPlum fragments are shown in FIG. 2. In addition, normalized absorption spectra (solid line) and fluorescence spectra (dashed line, excitation at 570 nm) of mPlum, mPlum E16V, CpmPlum E16V, mPlum fragments are shown in FIG. 3. In addition, the manufacturing process of the mPlum fragment having a self-binding activity according to the present invention is shown in FIG.

As shown in FIG. 2, the CpmPlum mutant (CpmPlum E16V) and co-expression of mPlum 2-11 and mPlum 1 E16V that had been circularly sequenced in E. coli emitted red fluorescent signals.

As also shown in FIG. 3, the purified protein showed a maximum emission at 615 nm, slightly blue shifted from the original protein. Blue shifts in fluorescence emission of Glu 16 mutants of mPlum have been previously reported [X. Shu, L. Wang, L. Colip, K. Kallio, SJ Remington, Protein Sci. 2009 , 18 , 460.]. Absorption spectra and fluorescence spectra of the protein complex (MBP-mPlum 1 E16V + His-mPlum 2-11) and the ordered CpmPlum mutant (CmPlum E16V) were indistinguishable. In addition, the spectroscopic properties of the semisynthetic bound protein (mPlum 1 E16V peptide + mPlum 2-11) were nearly identical to the spectroscopic properties of the co-expressed protein. Thus, it was confirmed that the mPlum 2-11 fragment self-bound with the co-expressed or synthesized mPlum 1 E16V peptide. That is, it can be seen that the mPlum 1 E16V peptide has self-binding activity.

Experimental Example 1  : In vitro Complementarity of mPlum Fragments

In vitro complementarity of mPlum fragments was performed using mPlum 2-11 and synthetic peptide mPlum 1 E16V (EVIKEFMRFKVHMEGSVN, Peptron). Final concentration of 1 μM of mPlum 2-11 protein and 1-4 μM of mPlum 1 E16V peptide in varying binding buffer (50 mM Tris pH 7.4, 0.1 M NaCl, 10% glycerol, 1% DMSO, and 0.2-0.8 M urea) Both fragments were mixed with. Binding and fluorescence complementarity of mPlum 2-11 with synthetic mPlum 1 E16V peptide was observed for 50 h at 570 nm excitation and 615 nm emission.

The results are shown in FIG.

As shown in FIG. 5, in vitro complementarity between 1 μM of mPlum 2-11 protein and 1-4 μM of mPlum 1 E16V peptide showed a higher concentration of mPlum 1 E16V peptide according to the change in concentration of urea (0.2-0.8 M). 4 μM) provided faster complementarity and reached equilibrium after approximately 24 hours.

Experimental Example 2  : Of mPlum 2-11 with Multiple mPlum 1 Peptide Mutants in vivo  Complementarity

In order to investigate the self-binding ability of various mPlum 1 peptide mutants and mPlum 2-11, the following experiment was performed. In other words, since the residues of the peptides affect fluorescence emission, Glu 16 of the mPlum 1 peptide can be converted into various compounds such as Gly (glycine), Ile (isoleucine), Met (methionine), Gln (glutamine), and Ser (serine). Mutated with amino acids. In vivo complementarity of several mPlum 1 peptide mutants with mPlum 2-11 was then examined by co-expression in E. coli .

The results are shown in FIG.

As shown in FIG. 6, the mutant (mPlum 1 E16V) replacing Glu 16 of mPlum 1 peptide with Val (valine) showed high levels of in vivo fluorescence complementarity, while Glu 16 of mPlum 1 peptide was expressed as Ile (iso). Mutants (mPlum 1 E16I, mPlum 1 E16M) replaced with leucine) or Met (methionine) showed low levels of in vivo fluorescence complementarity and the other mutants did not show fluorescence signals. Thus, it can be seen that the mPlum 1 E16V peptide has good self-binding activity.

Experimental Example 3  : Minimal residue investigation and fluorescence spectra of mPlum 1 E16V peptides for self-binding with mPlum 2-11

Minimum residues of the mPlum 1 E16V peptide for self-binding with mPlum 2-11 were investigated by systematically removing the N- or C-terminal amino acids of the peptide. The residues (amino acid sequence) of the mPlum 1 E16V peptide used in the experiment are shown in Table 2 below.

In addition, the fluorescence spectrum of mPlum 1 E16V C-1 peptide coupled with mPlum 2-11 and the fluorescence spectrum of CmPlum E16V coupled with mPlum 2-11 were compared and observed, and are shown in FIG. 7.

TABLE 2

Peptide name	Amino acid sequence	Self-coupling
Long mPlum
1 E16V	VSKGEEVIKEFMRFKVHMEGSVN	has exist
mPlum
1 E16V	EVIKEFMRFKVHMEGSVN	has exist
mPlum
1 E16V C-1	EVIKEFMRFKVHMEGSV	weakness
mPlum
1 E16V C-3	EVIKEFMRFKVHMEG	none
mPlum
1 E16V N-3	KEFMRFKVHMEGSVN	none
mPlum
1 E16V N-6	MRFKVHMEGSVN	none

As shown in Table 2 and FIG. 7, the minimum residues of the mPlum 1 E16V peptide for self-binding with mPlum 2-11 were 18 amino acids, which were confirmed to be the critical point of maximum self-binding. That is, it was confirmed that removing only one residue from the peptide mPlum 1 E16V having 18 amino acid sequences significantly interfered with fluorescence complementation.

Experimental Example 4  : Tagging and Detection of Protein-Peptide (mPlum 1 E16V) in Mammalian Cells

1. Fluorescence Imaging of HeLa Cells Expressing Multiple mPlum Fragments

We investigated whether mPlum 1 E16V peptides with self-binding activity can be used for tagging and detection of proteins in mammalian cells. Genetic-encoded peptide tagging and detection of peptides with externally applied fluorescent compounds overcomes many limitations of rather large fluorescent protein tags [a) I. Chen, AY Ting, Curr. Opin. Biotechnol. 2005 , 16 , 35; b) HM O'Hare, K. Johnsson, A. Gautier, Curr. Opin. Struct. Biol. 2007 , 17 , 488.]. However, the widespread use of such peptide-compound labeling methods has been limited by nonspecific basis signals, cell permeation, and limited resources of the compounds. Therefore, in this experiment, genetically encoded mPlum 2-11 protein was used to replace fluorescent compounds. Specifically, to express the protein in mammalian cells, the gene of the protein was cloned with the pcDNA expression vector (Invitrogen). Cervical cancer cell lines (HeLa cells) were transiently transformed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. After transformation, various forms of mPlum fragments were expressed in HeLa cells for 24 hours, and fluorescent images of HeLa cells with DeltaVision RT fluorescent microscope (Applied Precision) using 555/28 nm excitation filter and 617/23 nm emission filter. Got.

The results are shown in FIG.

As shown in FIG. 8, mPlum and CpmPlum E16V showed red fluorescent activity in HeLa cells, whereas mPlum 2-11 fragment alone did not show fluorescent activity. However, the co-transformers of mPlum 2-11 and MBP-mPlum 1 E16V peptides showed bright red fluorescence. Thus, it was confirmed that the mPlum 1 E16V peptide tag can be used for tagging and detection of proteins in mammalian cells (HeLa cells).

2. Fluorescence imaging of tetracycline-regulated HeLa cells that sequentially express several mPlum fragments

Sequential expression of mPlum fragments was performed with tetracycline-regulated expression (T-Rex) HeLa cell line (Invitrogen). Tetracycline-regulated expression (T-Rex) HeLa cells were grown according to the manufacturer's protocol and transformed with pcDNA4 / TO expression vectors containing genes of mPlum or mPlum 2-11 fragments. MBP-mPlum 1 E16V peptide was not regulated by tetracycline and thus cloned into pcDNA. On the first day, tetracycline-regulated expression (T-Rex) HeLa cells were co-transformed with pcDNA-MBP-peptide (mPlum 1 or mPlum 1 E16V peptide) and pcDNA4 / TO-mPlum 2-11. On the second day, tetracycline (1 μg / ml) was added to the cell culture to induce the expression of mPlum 2-11 protein. Fluorescence images of HeLa cells were obtained at various tetracycline induction time points. To confirm the expression of tetracycline-induced protein in HeLa cells, wild type mPlum protein was also induced by tetracycline.

Fluorescence images of tetracycline-regulated HeLa cells expressing several mPlum fragments sequentially are shown in FIG. 9, while fluorescent images of tetracycline-regulated HeLa cells sequentially expressing several mPlum fragments at the time of induction of tetracycline are shown in FIG. 10. Shown in

As shown in FIG. 9, MBP protein tagged in mPlum 1 E16V peptide not regulated by tetracycline was immediately expressed, whereas expression of mPlum 2-11 protein was inhibited by tetracycline regulation. In addition, wild-type mPlum protein showed a red fluorescence signal upon tetracycline induction. Thus, it can be seen that fluorescence labeling of MBP-mPlum 1 E16V peptide was successful by inducing mPlum 2-11 protein by addition of tetracycline.

In addition, as shown in FIG. 10, the red fluorescence signal of mPlum was clearly observed within 6 hours after tetracycline induction, indicating that mPlum 2-11 required 8 hours to bind to MBP-mPlum 1 E16V peptide and form a mature chromophore. Confirmed.

Experimental Example 5  : Characteristics of Synuclein-peptide Fusion Proteins

The α-syn gene tagged with the α-syn wild type and c-terminal mPlum 1 E16V was cloned into pET21a. BL21 (DE3) cells containing the above plasmids were incubated at 37 ° C. until the OD600 was 0.6, and the expression of the protein was induced by 1 mM IPTG after 3 hours. Expression induced cells were 10 mM Tris pH 7.4. Collection and expression were stopped.

The cell suspension was heated to 100 ° C. for 20 minutes and the supernatant collected using centrifugation. The supernatant was slowly cooled, loaded into a Q-Sepharose Fast Flow column (GE Healthcare), and dissolved using 10 mM Tris (pH 7.4) containing 200 mM NaCl. Purified α-syn protein was dialyzed with 20 mM HEPES pH 7.2 and 10 mM PMSF and stored at -80 ° C.

For in vitro assays, 500 mL of 80 mM protein containing α-syn sample in 20 mM HEPES (pH 7.2) and 10 mM PMSF was subjected to 37 ° C., continuous stirring conditions using a glass vial. Incubated at. Fibril formation formation was observed by fluorescence detection and transmission electron microscopy (TEM) analysis with 20 mM Thioflavin T after negative staining with 2% uranyl acetate and the results are shown in FIG. 11. .

Small mPlum E16V peptide (18 amino acids) was found to be fused to the C terminus of α-syn. The fibrosis properties of the peptide-tagged α-syn were confirmed to be similar to the α-syn wild type, as is the standard ThioFlavin T fluorescence assay and TEM analysis (FIGS. 11A and B). The exact effect of the large fluorescent protein tag (w27 kDa) on the basic aggregation properties is more controversial than the relatively small α-syn (w15 kDa). However, small tags were found to be more suitable for the study of α-syn folding and flocculation methods. More importantly, recent studies show that cell-to-cell delivery of α-syn is a key factor in pathology.

Peptide tag detection of α-syn may be valuable for cell-to-cell monitoring because of large proteins that significantly affect cellular protein penetration, such as GFPs. Moreover, multiple chains of self-assembly of fluorescent protein fragments may be possible through observation of various sources of α-syn protein in living cell systems. Here, in vitro and Escherichia coli, mPlum 2-11 and α-syn-tagged mPlum 1 E16V self-assembly and the generation of fluorescence signal was confirmed and shown in Figure 11 c and d.

Experimental Example 6  : Synuclein-peptide Detection in Mammalian Cells

For gene expression in mammalian cells, protein genes were cloned through pcDNA expression vectors (Invitrogen). HeLa cells were transformed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. Various forms of mPlum fragments were expressed 24 hours after infection in HeLa cells, and fluorescence images were obtained by DeltaVision RT fluorescent microscope (Applied Precision).

MNBP peptide-taking and α-syn protein were not regulated by the tetracycline of Experiment 2 above and cloned into pcDNA.

On the first day, tetracycline-regulated expression (T-Rex) HeLa cells were co-transformed with pcDNA-α-syn-peptide (mPlum 1 or mPlum 1 E16V peptide) and pcDNA4 / TO-mPlum 2-11. On the second day, tetracycline (1 μg / ml) was added to the cell culture to induce the expression of mPlum 2-11 protein. Fluorescence images of HeLa cells were obtained at various tetracycline induction time points. To confirm the expression of tetracycline-induced protein in HeLa cells, wild type mPlum protein was also induced by tetracycline.

Through immunohistochemical analysis, cell expression α-syn-peptides were fixed and permeated according to the manufacturer's manual (BD Bioscience). Cell nuclei were stained with Hoechst 33342, and various forms of α-syn were stained with α-syn antibody Syn211, which is shown in FIG. 12.

The α-syn-mPlum 1 E16V genetically encoded with mPlum 2-11 protein was observed. Peptide tagged α-syn was expressed for 1 day before mPlum 2-11 fluorescence detection. Cell α-syn proteins were imaged using both mPlum assembly and α-syn-specific antibodies.

As shown in FIG. 12, both homogeneously distributed fluorescence signals (possibly non-collective) and more localized signals (possibly collective) were correlated with mPlum assembly and antibody staining. Even in the coexistence of mPlum and antibody signals, there was a clear cell-to-cell change.

It has been reported that various α-syn detection tools and various sources of α-syn-specific antibodies provide high immunohistochemical results of cellular α-syn imaging. With self-assembled GFP and mPlum fragments, two different α-syn-peptide proteins could be observed. For example, both genetically expressed and exogenously delivered α-syn-peptide proteins may be monitored simultaneously. In addition, genetically encoded mPlum 2-11 fragments also allow detection of α-syn-peptide targets, which may be related to specific parts of the cell. Thus we are studying the effect of α-synpeptide folding state for mPlum complementarity.

The foregoing description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, the embodiments described above are to be understood in all respects as illustrative and not restrictive.

The red fluorescent protein fragment (mPlum 1 E16V) having self-binding activity according to the present invention can be used as a biosensor as a useful tool for analyzing protein functions such as protein location in living cells, protein-protein interactions, and the like. .

SEQ. ID. NO. 1 (amino acid sequence of mPlum 1 E16V peptide): EVIKEFMRFK VHMEGSVN

SEQ. ID. NO. 2 (amino acid sequence of mPlum): MVSKGEEVIK EFMRFKEHME GSVNGHEFEI EGEGEGRPYE GTQTARLKVT KGGPLPFAWD ILSPQIMYGS KAYVKHPADI PDYLKLSFPE GFKWERVMNF EDGGVVTVTQ DSSLQDGEFI YKVKVRGTNF PSDGPVMQKK TMGWEASSER MYPEDGALKG EMKMRLRLKD GGHYDAEVKT TYMAKKPVQL PGAYKTDIKL DITSHNEDYT IVEQYERAEG RHSTGA

SEQ. ID. NO. 3 (amino acid sequence of CpmPlum): MHEFEIEGEG EGRPYEGTQT ARLKVTKGGP LPFAWDILSP QIMYGSKAYV KHPADIPDYL KLSFPEGFKW ERVMNFEDGG VVTVTQDSSL QDGEFIYKVK VRGTNFPSDG PVMQKKTMGW EASSERMYPE DGALKGEMKM RLRLKDGGHY DAEVKTTYMA KKPVQLPGAY KTDIKLDITS HNEDYTIVEQ YERAEGRHST GAGGTGGSGG TGGSVSKGEE VIKEFMRFKE HMEGSVN

Claims (9)

1. Characterized by replacing the Glu (glutamic acid) of 16 of the amino acid sequence of β-strand 1 of the red fluorescent protein (mPlum) with Val (valine), red having self-binding activity having the amino acid sequence of SEQ ID NO: Fluorescent protein fragment (mPlum 1 E16V).
2. The gene encoding the red fluorescent protein fragment (mPlum 1 E16V) of claim 1.
3. An expression vector comprising the gene of claim 2.
4. A host cell comprising the expression vector of claim 3.
5. The host cell of claim 4, wherein the host cell is E. coli .
6. Claim 1 protein detection kit comprising a red fluorescent protein fragment (mPlum 1 E16V).
7. 1) preparing the ordered mPlum protein (CpmPlum) by connecting the β-strand 1 of the red fluorescent protein (mPlum) through the flow peptide linker behind the β-strand 2-11;

   2) preparing a mutant (CpmPlum E16V) by substituting Val (valine) for 16 Glu (glutamic acid) in the amino acid sequence of β-strand 1 of the ordered mPlum protein (CpmPlum),

   3) separating the mutant (CpmPlum E16V) into mPlum 2-11 fragment and mPlum 1 E16V fragment, and

   4) A method of producing a red fluorescent protein fragment (mPlum 1 E16V) having a self-binding activity of claim 1, comprising the step of self-binding the separated mPlum 2-11 fragment and mPlum 1 E16V fragment.
8. MPlum 1 E16V (MBP-mPlum 1 E16V) and His-tagged mPlum 2-11 (His-mPlum 2-11) fused with maltose-binding protein were expressed together in the host cell and then in the host cell. A method for the analysis of protein interaction using a red fluorescent protein fragment (mPlum 1 E16V) having self-binding activity of claim 1, characterized by detecting a red fluorescent signal.
9. The method of claim 8, wherein the host cell is E. coli . 10. The method of analyzing protein interaction using a red fluorescent protein fragment (mPlum 1 E16V) having self-binding activity.

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