KR100825290B1 - System of Biomolecular Fluorescence Complementation Containing a Cell Inserted a Part of Fluorescence Protein Gene In Chromosome and Methods of Biomolecular Fluorescence Complementation By Using It - Google Patents

Abstract

The present invention relates to a bimolecular fluorescence complementation technique for real-time analysis of intracellular protein interactions and a plasmid vector for applying the same. Unlike the conventional extracellular analysis or protein interaction analysis method through artificial protein expression, the bimolecular fluorescence complementation technique of the present invention provides a real-time interaction between the proteins expressed in their natural state from their own promoters in the cell in real time. Not only can it be analyzed, it can also analyze the intracellular locations where protein interactions occur.

Yeast, Bimolecular Fluorescence Complementary Technique, Fluorescent Protein, Protein Interaction, Intracellular Location

Description

System of Biomolecular Fluorescence Complementation Containing a Cell Inserted a Part of Fluorescence Protein Gene In Chromosome and Methods of Biomolecular Fluorescence Complementation By Using It}

1 is a schematic diagram of a bimolecular fluorescence complementary technique;

2 is a process for preparing a fluorescent protein fragment of the present invention and a vector for attaching the fluorescent protein fragment;

3 is a genetic map of the pFA6a-VN173-HIS3MX6 vector of the present invention;

4 is a genetic map of the pFA6a-VC155-HIS3MX6 vector of the present invention;

5 is a process for attaching a fluorescent protein fragment to a specific gene terminus;

6 is a result of real-time analysis of the interaction and location of the protein within the cell using a bi-molecule fluorescence complementary technique.

The present invention relates to a method for analyzing protein interactions in a cell in real time, and more particularly, to the development and analysis of a system for analyzing intracellular protein interactions using a bimolecular fluorescence complementation technique. will be.

All the phenomena happening in the cell are caused by proteins, in which the proteins function mainly as complexes, and one protein in the cell plays several roles by changing the combination of proteins that make up the complex. You can do it. Therefore, in order to study cells and other organisms, analysis of protein-protein interaction (protein-protein interaction) can be said to be indispensable.

As the importance of understanding protein interactions increases, experimental techniques for analyzing them have also been developed. A brief review of the protein interaction analysis methods developed to date includes immunoprecipitation as an experiment that is generally performed in the laboratory to examine protein interactions. Proteins in vivo perform their functions by binding to other proteins, so when one protein is purified, other proteins that bind to it are also purified. If Y is the other protein that binds X, then a compound precipitate of X / Y / X-antibody can be obtained when the antibody against X is added. If Y is detected after electrophoresis analysis of this precipitate, X-Y interaction 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. For example, Gavin et al . (Gavin et al ., Nature 2002, 415: 141-147) and Ho et al . (Ho et al ., Nature 415: 180-183) attach an affinity tag to hundreds of yeast proteins. The immunoprecipitated proteins were identified by mass spectrometry and recently analyzed the interaction of over 4000 proteins out of a total of 6000 yeast proteins (Gavin et al ., Nature 2006, 440: 631-636; Krogan et al ., Nature 2006, 440: 637-643).

Zhu and Snyder (Zhu & Snyder, Curr. Opin. Chem. Biol. 2003 7: 55-63) reported that protein chips can be of great help in analyzing protein interactions. 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 due to various parameter changes. Ptacek et al ., Nature 2005, 438: 679-684, reported protein interactions on whole proteins using protein chips in the budding yeast Saccharomyces cerevisiae .

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. Several systems have been developed to overcome the limitations of these in vitro experiments and to more accurately analyze protein interactions in vivo.

The yeast two-hybrid system, a recently used method, is a method that can analyze protein interactions through transcriptional activity of reporter genes in living yeast cells rather than extracellularly. It is widely used as a way to explore protein interactions in libraries (MATCHMAKER Two-Hybrid System User Manual, Clontech). The yeast two-hybrid system has the advantage of being able to see protein interactions in vivo, contributing to the study of protein interactions in cells of higher animals. In case of yeast-2 hybrid using GAL4, DNA binding region and transcriptional activation region of GAL4 transcription factor of yeast are separated and expressed, and GAL4 upstream activating sequence is located on the reporter gene (e.g. lacZ ) promoter. Many systems are built to exist. When the two proteins interacting with the system are expressed together with the DNA binding region and the transcriptional active region of GAL4, respectively, the protein expressed in one form with the DNA binding region of GAL4 binds to the GAL4 upper active sequence and simultaneously GAL4 transcription. The GAL4 transcriptional active region interacts with the expressed protein along with the active region, and finally interacts with RNA polymerase II to express the reporter gene lacZ . However, the yeast 2-hybrid system is useful for analyzing protein interactions inside the nucleus because it is a system of complementary binding of transcription factors. However, the yeast 2-hybrid system shows a positive error result when analyzing protein interactions outside the nucleus. .

Miyawaki et al . (Miyawaki et al ., Nature , 1997, 388: 882-887) use calmodulin and calmodulin-binding peptides using the fluorescence resonance energy transfer (FRET) principle. By analyzing the interactions between binding peptides, a new concept of protein interaction analysis system was developed. Fluorescent proteins are excited by absorbing light in different unique wavelength ranges, and their energy is restored to the ground state after the energy is emitted by light or heat, and each fluorescent protein emits a unique wavelength band of light. The fluorescence resonance energy transfer principle is that when two different fluorescent proteins are within 10 to 100 Hz, the light emitted after excitation of the short wavelength fluorescent protein induces the excitation of the long wavelength fluorescent protein to emit fluorescence. That is, by attaching two different fluorescent proteins to each of the two proteins to examine the interaction, and then detecting the 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. For example, a fluorescent protein excited by light of 488 nm wavelength emits fluorescence in the 520 nm wavelength range, which acts as a wavelength for stimulating another paired fluorescence protein and emits fluorescence in the 630 nm wavelength band. . Thus, the interaction between the two proteins can be analyzed by excitation at 488 nm and detection of fluorescence at the 630 nm wavelength. This system has the advantage of being able to analyze the dynamic interaction of proteins in living cells, but only when the distance between two fluoroproteins is very close to detect them, and overexpression of proteins is necessary to detect subtle changes in fluorescence wavelength. In addition, expensive equipment is required for fluorescence resonance energy transfer analysis.

Hu et al . ( Mol. Cell 2002, 9: 789-798) reported the possibility of protein interaction analysis using bimolecular fluorescence complementation in higher animal cells. The two-molecule fluorescence complementation technique applies a previously reported protein fragment complementation to a fluorescent protein, divides the fluorescent protein into N- and C-terminal fragments, and then expresses them together with the two proteins to be examined. Then, when two proteins are closer to each other for interaction, a method of analyzing the fluorescence that appears when two fragments of the fluorescent protein are combined to form an intact fluorescent protein ( FIG. 1 ). Hu et al. Divided the yellow fluorescent protein into the N- and C-terminals using the above system, and then attached them to the Fos and Jun proteins, which are transcription factors, to express them in higher animal cells. It has been reported that protein interactions are taking place and that the ZIP domain plays an important role in its binding. In addition, Hu et al. (Hu & Kerppola, Nat. Biotechnol. 2003, 21: 539-545) used the system to analyze complex protein interactions using fluorescent proteins of various colors in higher animal cells. Bracha et al . (Bracha et al ., Plant J. 2004, 40: 419-427) and Walter et al . (Walter et al ., Plant J. 2004, 40: 428-438) complied with bimolecular fluorescence in higher plant cells at similar times. Hoff et al . ( Curr. Genet. 2005, 47: 132-138) reported that the technique could be applied to the Acremonium chrysogenum , a kind of filamentous fungus. It has been reported that fluorescence complementation techniques can be applied. In addition, many recent results of protein interaction analysis using bi-molecule fluorescence complementation techniques have been reported, and the advantages of the analysis of protein interactions and the intracellular location of interactions in living cells are recognized.

However, the bi-molecular fluorescence complementation system mentioned above actually occurs in cells because it clones the gene sequence encoding the protein and the sequence encoding the fragment of the fluorescent protein in the plasmid and then artificially expresses to see the interaction of the target protein. There is a possibility that it may be a little different from the situation, and it is difficult to say that the protein interactions in the cell are exactly as they are. Accordingly, an object of the present invention is to implement a two-molecule fluorescence complementary system on a chromosome rather than a plasmid so that the target protein-fluorescent protein fragment is expressed from its own promoter in the cell, thereby real-time monitoring of intracellular protein interactions in a natural state. Is to develop a system that can In order to achieve the object of the present invention, the present inventors have identified a plasmid vector which can be directly attached to the C terminus of a target protein gene on a chromosome by PCR in a yeast cell to be expressed through a promoter specific to the target protein. It was developed and applied to confirm that real-time analysis of protein interactions in living cells is possible.

It is an object of the present invention to provide a plasmid vector necessary for analyzing intracellular protein interactions in real time using a bimolecular fluorescence complementary technique, and to provide a method for analyzing intracellular protein interactions in real time by applying the same.

In order to achieve the above object, the present invention provides diploid cells other than humans, each of which contains a part of the fluorescent protein at the C-terminus of specific genes of the chromosome rather than the gene in the plasmid. The diploid (2N) cells are formed by the crossing of two haploid (N) recombinant cells. The C-terminus of the specific gene A of a specific chromosome in one haploid recombinant cell is linked to the N-terminal part of the fluorescent protein, and the C-terminus of the fluorescent gene C-terminus of the specific gene B of the specific chromosome in another haploid recombinant cell. Some are included. Although only a part of the fluorescent protein linked to the specific gene of the haploid cell does not fluoresce, the diploid cell made by the hybridization of the haploid cell is the fluorescent protein linked to these proteins when the specific gene A and the specific gene B are expressed and fused. Each part is fused to fluoresce (see FIG. 1 ). As the fluorescent protein, Green Fluorescence Protein ( GFP ), Yellow Fluorescence Protein ( YFP ), etc. may be used.

Another aspect of the invention relates to a vector for preparing haploid cells forming the diploid. The vector is a linking peptide sequence that provides flexibility when attaching a yellow fluorescent protein fragment to the C terminus of a specific gene, a sequence encoding a yellow fluorescent protein N terminus or a C terminus fragment, and is involved in specific amino acid synthesis as a trophogenic selection gene. It is a vector containing a sequence encoding an enzyme. More preferably, the plasmid vector for attaching the yellow fluorescent protein N-terminal fragment having the gene map shown in FIG. 3 or the plasmid vector for attaching the yellow fluorescent protein C-terminal fragment having the genetic map shown in FIG. 4 is even more preferably the pFA6a of SEQ ID NO: 1. -VN173-HIS3MX6 vector or pFA6a-VC155-HIS3MX6 vector of SEQ ID NO: 2.

Another embodiment of the present invention provides a haploid cell transformed by the plasmid vector for fluorescent protein attachment. The origin of the haploid cells may be from eukaryotic cells, including animal cells, plant cells, and fungal cells. Preferred are fungal cells, more preferred yeast. Specifically, the haploid cells are yellow fluorescent protein with a genetic map according to yellow fluorescent protein N-terminal fragment mounting plasmid vector transformed by the haploid Saccharomyces as MY access celebrity on busy or figure 4 has a gene map as described in figure 3 C Haploid Saccharomyces cerevisiae transformed with a terminal fragment plasmid vector. More specifically, Saccharomyces cerevisiae transformed by a haploid Saccharomyces cerevisiae transformed by a pFA6a-VN173-HIS3MX6 vector of SEQ ID NO: 1 or by a pFA6a-VC155-HIS3MX6 vector of SEQ ID NO: 2 Ada.

Another embodiment of the present invention is a diploid cell formed by the crossing of said haploid cells. The diploid cell may be a fungal cell, preferably yeast.

Another aspect of the invention provides a method for real-time analysis of intracellular protein interactions using bimolecular fluorescence complementation techniques comprising the following steps.

(A) Transform a portion of the fluorescent protein N terminus at the end of a specific protein gene of the intracellular chromosome in the haploid state and exclude the portion of the N terminus at the end of a specific gene of the intracellular chromosome in another haploid state that crosses the cell in the haploid state Transforming a portion of the fluorescent protein C terminus;

(B) propagating the two transformed cells, respectively;

(C) selecting only diploids formed by crossing the proliferated cells; And

(D) observing the formed diploid with a fluorescence microscope

More specifically, the present invention provides a method for real-time analysis of intracellular protein interactions in Saccharomyces cerevisiae using two-molecule fluorescence complementation technique.

(A) Fluorescent protein at the end of a specific protein gene in the haploid yeast chromosome transforms a portion of the fluorescent protein N terminal, and a fluorescent protein excluding the portion of the N terminal at the end of a specific gene of another haploid yeast chromosome that crosses the yeast in the haploid state Transforming a portion of the C terminus;

(B) culturing the transformed Saccharomyces cerevisiae;

(C) crossing the transformed Saccharomyces cerevisiae to select a diploid; And

(D) observing the transformed diploid Saccharomyces cerevisiae with a fluorescence microscope

In the above method, a diploid selection method may use a plurality of selection markers, but most preferably, it is selected using a nutritional selection marker. However, it is not limited thereto. Those skilled in the art can select diploids using various selection markers.

The present invention provides a step of preparing a plasmid vector capable of attaching a fluorescent protein fragment to the C terminus of a target protein gene on a chromosome, transforming a yeast cell using a vector and PCR for attaching the fluorescent protein fragment, a fluorescent protein N terminal, and Comprising a step of mating the yeast cells transformed at the C terminus, and fluorescence microscopic analysis of diploid cells expressing the fluorescent protein N terminus and the C terminus together.

Hereinafter, examples will be described so that the present invention can be understood in more detail. However, the following examples are not intended to limit the gist of the present invention.

Example 1 Amplification of the N- and C-terminal Fragment Genes

The composition of the reaction solution used for PCR amplification was a DNA template (pBiFC-VN173 or pBiFC-VC155, provided by Professor Purdue University Hu), 500 nM primer, 2 mM dNTP, 10X Pfu PCR buffer, Pfu DNA polymerase, and the total volume was Adjust to 50 μl. PCR amplification conditions were 2 minutes 30 seconds (1 cycle) at 94 ° C, 30 seconds at 94 ° C, 30 seconds at 55 ° C and 2 minutes (36 cycles) at 72 ° C, and 10 minutes (1 cycle) at 72 ° C. . The PCR product was then electrophoresed at 100 V on a 1.5% agarose gel for 30 minutes to confirm that the 700 bp N protein fragment and the 500 bp C protein fragment were amplified.

Example 2 Preparation of Fluorescent Protein Fragment Attachment Vector

In order to process each PCR amplification amplified in Example 1 with restriction enzymes, a total of 90 μl of PCR amplification was purified using a PCR purification kit (Bionia, Korea) and eluted in 30 μl of H ₂ O. ). Then, a vector was produced in the same manner as in FIG. 2 . The PFA6a-GFP-HIS3MX6 (GenBank accession no. Also treated with PacI / AscI as in the fluorescent protein fragment gene. The PCR amplification and vector skeletons thus treated were linked at 4 ° C. for 12 hours using T4 DNA ligase to prepare pFA6a-VN173-HIS3MX6 and pFA6a-VC155-HIS3MX6, which are two-molecule fluorescent complementary attachment vectors. 3 and 4 show a gene map and a nucleotide sequence of a vector for attaching a fluorescent protein fragment prepared by the above method. 3 is a pFA6a-VN173-HIS3MX6 vector, which includes an amp sequence representing an ampicillin resistance sequence, an HIS3 sequence that is a histidine amino acid biosynthesis gene, and a fluorescent protein N-terminal sequence. 4 is a pFA6a-VC155-HIS3MX6 vector, which includes an amp sequence representing an ampicillin resistance sequence, a HIS3 sequence that is a histidine amino acid biosynthesis gene, and a fluorescent protein C-terminal sequence. The recombinant vector was transformed into E. coli (TG1), cultured in ampicillin selection medium, and then plasmid DNA was obtained from the grown colonies to confirm that the vector was properly constructed by restriction enzyme treatment.

Example 3: Transformation of yeast cells (ATCC 201388 and ATCC 201389)

Yeasts used for transformation were Saccharomyces cerevisiae ATCC 201388 ( MAT a his3 leu2 met15 ura3 ) and ATCC 201389 ( MAT α his3 leu2 lys2 ura3 ). In order to attach the fluorescent protein N- and C-terminals to the ATCC 201388 and ATCC 201389 strains, a yeast transformation method as shown in FIG. 5 was used. Once the fluorescent protein fragment attachment plasmids (pFA6a-VN173-HIS3MX6 and pFA6a-VC155-HIS3MX6) were PCR amplified with a template. The composition of the reaction solution used for PCR amplification was 5 μl of DNA template, 5 μl of two primers of 5 μM, 5 μl of dNTP of 2 mM, 5 μl of 10X Pfu PCR buffer, 0.5 μl of Pfu DNA polymerase, and 50 μl of total volume. Adjusted to. The cycle conditions for PCR amplification were 2 minutes 30 seconds (1 cycle) at 94 ° C, 30 seconds at 94 ° C, 30 seconds at 55 ° C and 2 minutes (36 cycles) at 72 ° C, and 10 minutes (1 cycle) at 72 ° C. Was. The PCR product was then electrophoresed at 100 V for 20 minutes on a 1.5% agarose gel to confirm that about 2 kb of the fluorescent protein N and the fluorescent protein C terminal fragments containing the HIS3 gene were amplified. Meanwhile, the yeast cells grown to OD ₆₀₀ = 1.0 in YPD medium (1% yeast extract, 2% peptone, 2% glucose) were recovered by centrifugation at 3000 rpm for 5 minutes, and then 0.1M lithium acetate 3 After the addition of ml and washing, the cells were again suspended in 300 [mu] L of 0.1 M lithium acetate. Then add 100 μl 50% (w / v) PEG 3350, 15 μl 1 M lithium acetate, 20 μl 4 mg / ml carrier DNA and 15 μl of the fluorescent protein fragment PCR amplification prepared above and mix well Then, incubated for 30 minutes at 30 ℃, 15 minutes at 42 ℃ using a PCR machine. The cells were recovered by centrifuging the cultured cells at 6000 rpm for 30 seconds, and then suspended in 100 μl of H ₂ O, and then divided into histidine deficient medium (SC-His; synthetic complete His and histidine 30). Incubated at about 3 days. The colonies grown on the selection medium were transferred to histidine deficient medium once again and cultured, and then the yeast colony PCR method was applied to the target genes by using the 500 bp upper sequence and the starting sequence of the fluorescent protein attachment vector at the end of the target gene as primers. It was confirmed that the fluorescent protein fragments were properly attached. Yeast colony PCR method is as follows. 20 μl of H ₂ O was scraped and suspended with a tip of cells grown in histidine deficient medium, and boiled at 100 ° C. for 15 minutes using a PCR machine. Then, 5 μl of the boiled cells, 2.5 μl of two primers of 5 μM and 2 mM PCR was performed with 1.5 μl of each dNTP, 2.5 μl of 10 × Taq PCR buffer, 0.5 μl of Taq DNA polymerase, and 25 μl total volume. Cycle conditions for PCR amplification were 3 minutes (1 cycle) at 94 ° C, 30 seconds at 94 ° C, 30 seconds at 55 ° C and 1 minute (36 cycles) at 72 ° C, and 10 minutes (1 cycle) at 72 ° C. Then, the PCR product was electrophoresed for 25 minutes at 100 V on a 1.5% agarose gel to confirm whether the DNA of about 500 bp was amplified.

Example 4: Cross-linking Yeasts Transfected with N- and C-Terminal Fluorescent Proteins

Saccharomyces cerevisiae yeast used in the experiment ATCC 201388 ( MAT a his3 leu2 met15 ura3 ) is histidine, leucine, methionine, uracil nutritional strain and ATCC 201389 ( MAT α his3 leu2 lys2 ura3 ) are histidine, leucine, lysine, and uracil nutrients strains.When two strains are crossed, methionine and lysine nutrients strains become diploids in methionine- and lysine-deficient media. diploid) strains can be screened. As shown in Figure 5 , ATCC 201388 transformed the fluorescence protein N terminus with the HIS3 marker, and ATCC 201389 transformed the fluorescent protein C terminus with the HIS3 marker. The two host cells transformed with the N- and C-terminus of the fluorescent protein can be crossed because they have different mating types, and methionine-lysine deficient medium (SC-Met-Lys; Met is methionine, Lys is lysine). ), Yeast strains with diploid numbers expressing both the N- and C-terminal fluorescent proteins were selected. The breeding process is as follows. In this manner, the ATCC 201388 and ATCC 201389 strains transformed with the N- and C-terminal fragments of the fluorescent protein were cultured in 5 ml of YPD medium using an incubator at 30 ° C. to OD ₆₀₀ = 1.0. Thereafter, 500 µl of each strain was mixed in a 1.5 ml tube, mixed, incubated for 2 hours or more at room temperature, and allowed to cross, cultured in methionine-lysine deficient medium for selection of hybridized diploid strains, and cultured for 3 days. It was.

Example 5: Protein Interaction Analysis by Microscopy

Preparation of the strain for fluorescence microscopy was performed in two ways. Tip of the colonies incubated at 30 ° C. for 15 hours in methionine-lysine deficient glucose medium, and then suspended in 200 μl of sterile H ₂ O, and then observing a certain amount by fluorescence microscopy, or SC (synthetic complete) liquid medium A method of observing a cell cultured at 30 ° C. at an OD _{600 of} 0.7 by fluorescence microscopy was performed. Microscopic analysis was performed using Carl Zeiss Axiovert 200M fluorescent inverted microscope equipped with a black and white CCD camera. In order to detect the fluorescence emitted by the complementary binding of the yellow fluorescent protein N- and C-terminal fragments by protein interaction, light of 450-490 nm wavelength was exposed using a Carl Zeiss B10 shift-free Blue broadband BP filter. Light of 515-565 nm wavelength was detected and the exposure time was set to 2000 ms.

Example 6 Analysis of Protein Interaction Using Transformed Yeast of the Present Invention (Sis1 Protein Interaction Analysis)

Sis1 is a protein involved in the structure of proteins in cells and is known to form a homodimer (Lu et al ., J. Biol. Chem. 273: 27824-27830). The above embodiment the fluorescent protein N-terminal fragment at the end of the SIS1 gene of ATCC 201388 cells 1 from over the course of up to 4, the end of the SIS1 gene of ATCC 201389 cells after creating the strain was deposited with the fluorescent protein C-terminal fragment , Example 4 to prepare a diploid yeast strain that crossed the two strains to observe the fluorescence microscope. (A) of FIG. 6 is a diploid yeast strain made by attaching a yellow fluorescent protein (YFP) to the end of the SIS1 gene of the ATCC 201388 strain as a positive control and crossing it with the ATCC 201389 strain. As a result of using a Carl Zeiss B10 shift-free Blue wide band BP filter, the intracellular location of the Sis1 protein was found to be evenly distributed throughout the cell, especially in the nucleus. On the other hand (b) of Figure 6 is a result of observing the fluorescence microscope of the strain to which the two-molecule fluorescence complementary technique was applied under the same conditions as the positive control. When the strains for the two-molecule fluorescence complementation technique were compared with the positive control group, Sis1 showed two-molecule fluorescence complementation by the interaction between the Sis1 proteins. I knew it existed. Figure 6 ( c) is a negative control (negative control) the result of observing the fluorescence microscope by making a diploid yeast strain having only one segment of the fluorescent protein N- or C-terminal fragments. In addition, the fluorescent protein fragments in the Cet1 protein at the same intracellular location as Sis1 are used to examine whether the two-molecule fluorescence complementation is due to the close proximity of the location of the fluorescent protein fragments in the cell and not by direct interactions between the Sis1 proteins. We attached and analyzed the interaction with Sis1. Negative control results show that there is almost no fluorescence when only one of the fluoroprotein fragments is present, and that the two-molecular fluorescence complementation is specifically caused by the actual protein interactions, not by accidental interactions between two proteins in the same location in the cell. And it was found.

The present invention provides a method for analyzing protein interactions with plasmid vectors for real time analysis of intracellular protein interactions using bimolecular fluorescence complementarity techniques. In contrast to the method of extracellular analysis or artificial protein expression using plasmids, the bimolecular fluorescence complementation technique of the present invention allows the protein to be expressed by its own promoter in the cell, thereby interacting with the protein in the natural state and intracellularly. Not only can the location be analyzed in real time, but also can be easily observed with a fluorescence microscope, so it can be very useful for intracellular protein interaction analysis.

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delete delete delete delete delete delete PFA6a-VN173-HIS3MX6 vector having SEQ ID NO: 1. delete PFA6a-VC155-HIS3MX6 vector having SEQ ID NO: 2. delete delete delete Real-time analysis method of intracellular protein interaction using bimolecular fluorescence complementary technique comprising the following steps. (A) Transform a portion of the fluorescent protein N terminus at the end of a specific protein gene of the intracellular chromosome in the haploid state and exclude the portion of the N terminus at the end of a specific gene of the intracellular chromosome in another haploid state that crosses the cell in the haploid state Transforming a portion of the fluorescent protein C terminus; (B) propagating the two transformed cells, respectively; (C) selecting only diploids formed by crossing the proliferated cells; And (D) observing the formed diploid with a fluorescence microscope A method for real-time analysis of intracellular protein interactions in Saccharomyces cerevisiae using two-molecule fluorescence complementation technique comprising the following steps. (A) Saccharomyces cerevisiae in a haploid state transforms a portion of the fluorescent protein N terminus at the end of a specific protein gene in the chromosome and crosses with the haploid Saccharomyces cerevisiae in another haploid state Transforming the mises cerevisiae with a portion of the fluorescent protein C terminus except for the portion of the N terminus at the end of a specific gene of the chromosome; (B) culturing the transformed Saccharomyces cerevisiae; (C) crossing the transformed Saccharomyces cerevisiae to select a diploid; And (D) observing the transformed diploid Saccharomyces cerevisiae with a fluorescence microscope

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