KR101646597B1 - PTEN Fusion Protein and Use Thereof - Google Patents

PTEN Fusion Protein and Use Thereof Download PDF

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KR101646597B1
KR101646597B1 KR1020150078549A KR20150078549A KR101646597B1 KR 101646597 B1 KR101646597 B1 KR 101646597B1 KR 1020150078549 A KR1020150078549 A KR 1020150078549A KR 20150078549 A KR20150078549 A KR 20150078549A KR 101646597 B1 KR101646597 B1 KR 101646597B1
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서병창
김동일
권혜진
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재단법인대구경북과학기술원
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Abstract

The present invention relates to a fusion protein of PTEN (phosphatase and tensin homolog) and a use thereof, and relates to a CFP-FKBP protein consisting of a fluorescent protein of the present invention and FKBP (FK506-binding protein) and a phosphatase Phosphatase domain (PD); A C2 domain (C2); The PTZ (phosphatase and tensin homolog) fusion protein composed of the C-terminal PDZ binding domain (PDZ-BD) was effectively used as a cell membrane by chemically-inducible dimerization and then PI (3,4 , 5) It was confirmed that P3 was selectively dephosphorylated. Therefore, the PTEN fusion protein can study not only intracellular biological phenomena by direct regulation of PI (3,4,5) P3 but also PTEN mutation or PI (3,4,5) P3, which is induced by overexpression of P3.

Description

[0001] PTEN Fusion Protein and Use Thereof [0002]

The present invention relates to PTEN (Phosphatase and Tensin homolog) fusion proteins and uses thereof, and more particularly to a phosphatase and tensin homolog (PTEN) fusion protein and a use thereof. More particularly, the present invention relates to a phosphatase and tensin homolog domain; PD); A C2 domain (C2); A PTEN fusion protein consisting of a PTEN protein comprising a C-terminal PDZ binding domain (PDZ-BD), a recombinant gene encoding said fusion protein, and a recombinant vector for expressing said fusion protein. In addition, the PTEN fusion protein is capable of regulating selective dephosphorylation of PI (3,4,5) P3 and can be utilized in intracellular biological phenomenon research.

PTEN is also called MTAC1 (mutated in multiple advanced cancers 1) or TEP1 (TGF regulated and epithelial cell enriched phosphatase 1). PTEN is composed of 403 amino acids and has a molecular weight of about 47,000 And is known to be expressed in many cell lines.

PTEN has the function of a dual specificity protein phosphatase with serine / threonine phosphatase and tyrosine phosphatase activity and also has phosphoinositide 3-phosphatase and It is also known to be capable of functioning as a lipid phosphatase that functions as inositol phosphate 3-phosphatase which dephosphorylates phosphoinositide 3 (PI3).

In addition, PTEN is known to inhibit tumors in response to PI3 phosphorylated enzyme-mediated amplification signal transduction cascades. Recently, however, PTEN has been shown to inhibit the dephosphorylation activity of protein (lipid) substrates independently of PI3 dephosphorylation activity And has been reported to interact with the tumor antigen p53 (Freeman DJ et < RTI ID = 0.0 > al ., Cancer Cell , 3: 117, 2003).

In other words, for the production of PI (4,5) P2 (phosphatidylinositol-4,5 bisphosphate), dephosphorylation activity using phosphatidylinositol-3,4,5 trisphosphate (PI) Has a protein dephosphorylation activity that dephosphorylates the phosphorylated tyrosine residue (Myers et al ., Proc Natl Acad Sci. , 94: 9052,1997). Due to these various anti-tumor activities, genetic mutants of PTEN induce various tumors and are performed by antagonizing the anti-apoptotic activity of the PI3K-Akt signaling pathway. These functions are known to regulate cell proliferation, cell survival, cell migration, and cell invasion, thereby allowing PTEN to function as a tumor suppressor (Besson A meat al ., Eur . J. Biochem . , 263: 605, 1990).

With regard to the therapeutic application of PTEN, WO 99/02704 discloses a method of treating hyperproliferative diseases such as prostate cancer and breast cancer using nucleic acid molecules encoding PTEN protein and PTEN, Patent No. 6,020,199 discloses an antisense oligonucleotide targeted to a nucleic acid encoding PTEN to control the expression of PTEN and a method of using the same to treat diseases associated with PTEN expression, for example, diabetes and hyperproliferative diseases.

On the other hand, dephosphorylation of PI (3,4,5) P3 activates various signal transduction systems within the cell. The most well-known downstream component is AKT, which is known to play a pivotal role in cell survival and growth. In addition, PI (3,4,5) P3 in the nervous system is known to be an important factor controlling the growth cone guidance and axon formation of neurons.

Conventional techniques have been developed for various types of chemically-inducible dimerization that can selectively dephosphorylate PI (4) P and PI (4,5) P2, another type of cell membrane phospholipid, , Especially ion channel studies and signal transduction mechanisms (Li T et al ., J Biol Chem. , 287 (48): 40680, 2012), an artificial protein transfer technique capable of selectively dephosphorylating PI (3,4,5) P3 has not yet been developed.

Thus, the present inventors have made intensive efforts to produce a recombinant fusion protein for selectively dephosphorylating PI (3,4,5) P3. As a result, PTEN (3,4,5) P3 (3, 4, 5) P3 in the cell membrane was selectively dephosphorylated to produce PI (3, 4, 5) 5) It was confirmed that the amount of P3 can be controlled, and the present invention was completed.

It is an object of the present invention to provide a PTEN fusion protein, a recombinant gene encoding the fusion protein, a recombinant vector for expressing the fusion protein, and a method for producing a PTEN fusion protein using the recombinant vector.

It is another object of the present invention to provide a method for dephosphorylating PI (3,4,5) P3 using the PTEN fusion protein.

In order to accomplish the above object, the present invention provides a method for producing a protein, comprising: i) a fluorescent protein; Ii) FKBP (FK506-binding protein) represented by the amino acid sequence of SEQ ID NO: 2; And iii) a phosphatase domain (PD), a C2 domain (C2), and a C-terminal PDZ binding domain (PDZ-BD) represented by the amino acid sequence of SEQ ID NO: Lt; RTI ID = 0.0 > PTEN < / RTI > fusion protein.

According to a preferred embodiment of the present invention, the fluorescent protein may be a green fluorescent protein (GFP), a cyan fluorescent protein (CFP), or a yellow fluorescent protein (YFP).

According to another preferred embodiment of the present invention, when the fluorescent protein is cyan fluorescent protein (CFP), the PTEN fusion protein may be represented by the amino acid sequence of SEQ ID NO: 1.

The present invention also provides a recombinant vector comprising the nucleotide sequence encoding the PTEN fusion protein, wherein the nucleotide sequence is represented by SEQ ID NO: 4, and a recombinant vector containing the recombinant gene.

The present invention also provides a method for producing a recombinant cell, comprising the steps of: preparing a recombinant cell which encodes a PTEN fusion protein or a recombinant vector containing the recombinant gene introduced into a host cell; Culturing the recombinant cell to produce a PTEN fusion protein; And recovering the resultant PTEN fusion protein.

Furthermore, the present invention provides a method for regulating the phosphatidylinositol-3,4,5 trisphosphate (PI (3,4,5) P3) dephosphorylation using the PTEN fusion protein .

According to a preferred embodiment of the present invention, the method for dephosphorylation of PI (3,4,5) P3 comprises: (a) a) a recombinant vector encoding the PTEN fusion protein or a recombinant vector containing the recombinant gene; And ii) a recombinant vector encoding a Lyn-FRB (FKBP-rapamycin binding domain) fusion protein represented by the amino acid sequence of SEQ ID NO: 5 or a recombinant vector containing the recombinant gene; Expressing the PTEN fusion protein and the Lyn-FRB fusion protein; (b) treating the cell with rapamycin to transfer the PTEN fusion protein to the cell membrane; And (c) inducing the dephosphorylation of PI (3,4,5) P3 by the PTEN fusion protein transferred to the cell membrane.

According to another preferred embodiment of the present invention, the Lyn-linker-FRB fusion protein expressed in step (a) is a cell membrane target protein, and the Lyn moiety may be present bound to the cell membrane.

According to another preferred embodiment of the present invention, the step (b) is a step of culturing the FRB of the Lyn-FRB fusion protein; Rapamycin; The binding of FKBP of the PTEN fusion protein may result in the PTEN fusion protein being transferred to the cell membrane.

The phosphatase domain (PD) which is a part of the PTEN protein and the C-terminus of the CFP-FKBP protein composed of the fluorescent protein of the present invention and FKBP (FK506-binding protein); A C2 domain (C2); The PTZ (phosphatase and tensin homolog) fusion protein composed of the C-terminal PDZ binding domain (PDZ-BD) was effectively used as a cell membrane by chemically-inducible dimerization and then PI (3,4 , 5) It was confirmed that P3 was selectively dephosphorylated. Therefore, the PTEN fusion protein can study not only intracellular biological phenomena by direct regulation of PI (3,4,5) P3 but also PTEN mutation or PI (3,4,5) P3, which is induced by overexpression of P3.

FIG. 1 is a schematic diagram showing the structure of the PTEN fusion protein (CF-PTEN) of the present invention and cell membrane migration of PTEN fusion protein by rapamycin. The red bar at the C-terminus of PTEN refers to the PDZ-binding domain. The PTEN fusion protein migrates to an anchored cell membrane by dimerization of FRB and FKBP by addition of rapamycin. The PD domain of CF-PTEN specifically binds PI (3,4,5) P3 Is dephosphorylated to PI (4,5) P2.
FIG. 2A shows confocal images of LDR, CF-PTEN and Btk-PH-GFP expressed in cells before and after treatment with 1 μM rapamycin for 120 seconds, with a scale bar of 5 μm . Fig. 2B shows the fluorescence intensity of cytosol. Blue indicates CF-PTEN and green indicates fluorescence intensity of Btk-PH-GFP.
3 shows the sequence of the PTEN fusion protein represented by the amino acid sequence of SEQ ID NO: 1 of the present invention.
Fig. 4 shows the sequence of a gene coding for the PTEN fusion protein of the present invention and represented by the nucleotide sequence of SEQ ID NO: 4.

Hereinafter, the present invention will be described in more detail.

As described above, the prior art has shown that various kinds of artificial protein transfer techniques (chemically-inducible dimerization) capable of selectively dephosphorylating PI (4) P and PI (4,5) P2, Has been actively used in the field of biology, especially ion channel studies and signal transduction mechanisms (Li T et al ., J Biol Chem , 287 (48): 40680, 2012), an artificial protein transfer technology capable of selectively dephosphorylating PI (3,4,5) P3 has not been developed yet.

Thus, the present invention sought to solve the above-mentioned problem by providing a PTEN (phosphatase and tensin homolog) fusion protein. When the PTEN fusion protein PTEN fusion protein is expressed in cells, the amount of PI (3,4,5) P3 in the cell can be controlled by selectively dephosphorylating PI (3,4,5) P3 present in the cell membrane, Fusion proteins not only can study intracellular biological phenomena by direct regulation of PI (3,4,5) P3, but also can induce mutations in PTEN or cancers induced by overexpression of PI (3,4,5) P3 And the like.

Accordingly, the present invention provides a method for producing a protein comprising the steps of: i) a fluorescent protein;

Ii) FKBP (FK506-binding protein) represented by the amino acid sequence of SEQ ID NO: 2; And

Iii) a phosphatase domain (PD), a C2 domain (C2), and a C-terminal tail PDZ binding domain (PDZ-BD) represented by the amino acid sequence of SEQ ID NO: PTEN protein;

(Phosphatase and tensin homologue) fusion protein.

The PTEN (phosphatase and tensin homologue or phosphatase and tensin homologue deleted from chromosome 10) is also referred to as MMAC1 (mutated in multiple advanced cancers 1) or TEP1 (TGF regulated and epithelial cell enriched phosphatase 1). PTEN is composed of 403 amino acids and has a molecular weight of about 47,000 Da and is known to have substrate specificity for PI (3,4,5) P3.

PTEN is a specific example of PI (3,4,5) P3, and it is known that D3 of phosphatidylinositol (PM) (Phosphatidylinositol-4,5 bisphosphate) is produced by dephosphorylation of phosphoric acid.

[Chemical Formula 1]

Figure 112015053614830-pat00001

In general, PTEN is a phosphoinositide-binding motif (PBM), a phosphatase domain (PD), a phosphatidylinositol (PM) bond for phosphatidylinositol The C2 domain (C2) and the C-terminal PDZ binding domain (PDZ-BD) which bind to phosphatidylserine (PS) of dillinositol.

In the present invention, a chemically-inducible dimerization system (CID system) was used to transfer PTEN protein to a cell membrane in which PI (3,4,5) P3 was present, and a fluorescent protein and FKBP (FK506-binding protein ) And a PTEN fusion protein containing PD, C2 domain and PDZ binding domain except for PBM (FIG. 1).

The artificial protein transfer technique utilizes the two proteins, FKBP and FRB (FKBP-rapamycin binding domain), as a characteristic of dimerization by the chemical rapamycin, Technology.

1, PTEN fusion protein (CF-PTEN) fused with Lyn-FRB (or Lyn-linker-FRB (LDR)) and CFP-FKBP was used in the present invention, and Lyn-linker-FRB ), Lyn is present in association with the cell membrane bound. When rapamycin is treated on cells expressing Lyn-linker-FRB and PTEN fusion proteins, the PTEN fusion protein is transferred to an anchored cell membrane by FRB-rapamycin-FKBP binding, and the PD domain of CF-PTEN Specifically phosphorylates PI (3,4,5) P3 to PI (4,5) P2.

In the present invention, the fluorescent protein may be a green fluorescent protein (GFP), a cyan fluorescent protein (CFP), or a yellow fluorescent protein (YFP) May be used without limitation. In the present invention, CFP is preferably used, and CFP; FKBP (FK506-binding protein); And a PTEN fusion protein composed of a PTEN protein including a phosphatase domain (PD), a C2 domain (C2), and a C-terminal tail PDZ binding domain (PDZ-BD) 1. ≪ / RTI >

In addition, the present invention relates to a recombinant vector encoding the PTEN fusion protein and a recombinant vector containing the recombinant gene, which is represented by the nucleotide sequence of SEQ ID NO: 4.

In one embodiment of the present invention, a DNA fragment coding for the 22 to 402 amino acid portion corresponding to PD, C2, and PDZ-BD, except for PBM, is amplified from a gene encoding PTEN protein to produce PTEN fusion protein, And fused with the gene encoding the CFP-FKBP protein to prepare a recombinant gene encoding the PTEN fusion protein represented by the nucleotide sequence of SEQ ID NO: 4.

Also, the present invention provides a method for producing a recombinant cell, comprising the steps of: preparing a recombinant cell which encodes a PTEN fusion protein or a recombinant vector containing the recombinant gene introduced into a host cell; Culturing the recombinant cell to produce a PTEN fusion protein; And recovering the resultant PTEN fusion protein.

The PTEN fusion protein prepared by the above method can be purified through various methods known in the art and can be purified using affinity chromatography, ion exchange chromatography, size exclusion chromatography, or the like.

The culturing and recovery of the recombinant cells for producing the PTEN fusion protein of the present invention can be applied to any culture method and separation and purification method of recombinant proteins conventionally known in the art without any particular limitation.

In the present invention, "vector" means a DNA product containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing the DNA in an appropriate host. The vector may be a plasmid, phage particle or simply a potential genome insert. Once transformed into the appropriate host, the vector may replicate and function independently of the host genome, or, in some cases, integrate into the genome itself. As the plasmid is the most commonly used form of the current vector, the terms "plasmid" and "vector" are sometimes used interchangeably in the context of the present invention. For the purpose of the present invention, it is preferable to use a plasmid vector. Typical plasmid vectors that can be used for this purpose include (a) a cloning start point that allows replication to be efficiently made to include several hundred plasmid vectors per host cell, (b) a host cell transformed with the plasmid vector And (c) a restriction enzyme cleavage site into which the foreign DNA fragment can be inserted. Even if an appropriate restriction enzyme cleavage site is not present, using a synthetic oligonucleotide adapter or a linker according to a conventional method can easily ligate the vector and the foreign DNA.

Preferably, the vector includes, but is not limited to, a plasmid vector, a cosmid vector, a bacteriophage vector, and a viral vector. Suitable expression vectors include signal sequence or leader sequences for membrane targeting or secretion in addition to expression control elements such as promoter, operator, initiation codon, termination codon, polyadenylation signal and enhancer, and may be prepared in various ways depending on the purpose. The promoter of the vector may be constitutive or inducible.

The recombinant cell according to the present invention can be produced by inserting the gene on a chromosome of a cell according to a conventional method, or introducing the recombinant vector onto a plasmid of a microorganism. After ligation, the vector should be transformed into the appropriate host cell. Generally preferred host cells are prokaryotic cells.

As a method for inserting the gene on the chromosome of the host cell, a commonly known gene manipulation method can be used in the present invention. For example, microinjection (direct insertion of DNA into cells), liposome, directed DNA uptake, receptor ~ mediated DNA transfer, or DNA transport using Ca ++ are some of the physical methods. Recently, viruses Gene transfer methods are widely used. Examples include retrovirus vectors, adenovirus vectors, adeno-associated viral vectors, herpes simplex virus vectors, poxvirus vectors, or lentiviral vectors. Particularly, retroviruses have high gene transfer efficiency, It can be used in a wide range of cells without binding by host DNA and rearrangement (resulting in alteration of host DNA function by altering the region of the host DNA similar to that of the host DNA).

A nucleic acid is "operably linked" when placed in a functional relationship with another nucleic acid sequence. This may be the gene and regulatory sequence (s) linked in such a way as to enable gene expression when a suitable molecule (e. G., Transcriptional activator protein) is attached to the regulatory sequence (s). For example, DNA for a pre-sequence or secretory leader is operably linked to DNA for a polypeptide when expressed as a whole protein participating in the secretion of the polypeptide; A promoter or enhancer may be operably linked to a coding sequence if it affects the transcription of the sequence; Or the ribosome binding site is operably linked to a coding sequence if it affects the transcription of the sequence; Or a ribosome binding site is operably linked to a coding sequence if positioned to facilitate translation. Generally, "operably linked" means that the linked DNA sequences are in contact and, in the case of a secretory leader, are in contact and present in the reading frame. However, the enhancer need not be in contact. The linkage of these sequences is carried out by ligation (linkage) at convenient restriction sites. If such a site does not exist, a synthetic oligonucleotide adapter or a linker according to a conventional method is used.

Furthermore, the present invention relates to a method for dephosphorylation of phosphatidylinositol-3,4,5 trisphosphate (PI (3,4,5) P3) using the PTEN fusion protein.

Specifically, the method for dephosphorylating PI (3,4,5)

(a) a) a recombinant vector encoding a PTEN fusion protein or a recombinant vector containing the recombinant gene; And ii) a recombinant vector encoding a Lyn-FRB fusion protein represented by the amino acid sequence of SEQ ID NO: 5, or a recombinant vector containing the recombinant gene, to produce a PTEN fusion protein and a cell membrane target protein Expressing the Lyn-FRB fusion protein;

(b) treating the cell with rapamycin to transfer the PTEN fusion protein to the cell membrane; And

(c) inducing the dephosphorylation of PI (3,4,5) P3 by the PTEN fusion protein transferred to the cell membrane;

. ≪ / RTI >

The recombinant gene encoding the PTEN fusion protein in step (a) is represented by the nucleotide sequence of SEQ ID NO: 4, and the recombinant gene encoding the Lyn-FRB fusion protein comprises the nucleotide sequence of SEQ ID NO: 6 .

The step (b) is based on an artificial protein transfer technique. As shown in FIG. 1, when the rapamycin treatment is performed, the FRB of the Lyn-linker-FRB fusion protein; Rapamycin; The binding of FKBP of the PTEN fusion protein causes the PTEN fusion protein to migrate to the cell membrane.

In one embodiment of the present invention, TsA201 cells derived from human embryonic kidney 293 cells ( 293 tsA1609neo ) stably transfected with SV40 T-antigen are mixed with a recombinant gene encoding the PTEN fusion protein of the present invention and a Lyn-linker A vector containing a recombinant gene encoding a -FRB (LDR) fusion protein was transformed to express PTEN fusion protein and Lyn-linker-FRB in cells, treated with rapamycin, and observed under a blue light microscope.

As shown in FIG. 2, it was confirmed that CF-PTEN migrates to an anchored cell membrane by dimerization of FRB and FKBP by addition of rapamycin. The PD domain of CF-PTEN is specific It was confirmed that PI (3,4,5) P3 was dephosphorylated with PI (4,5) P2. This can be confirmed by the movement of Btk-PH-, a biosensor of PI (3,4,5) P3. Also, as shown in the cytosol fluorescence intensity of FIG. 2B, it was confirmed that the addition of rapamycin gradually decreased the anti-CF-PTEN in which the cytosol fluorescence intensity of Btk-PH was rapidly increased.

That is, it was confirmed that the PTEN fusion protein of the present invention was used as a cell membrane by chemically-inducible dimerization and then selectively dephosphorylated PI (3,4,5) P3. Thus, the PTEN fusion protein It was confirmed that the intracellular biological phenomena can be studied by the direct regulation of PI (3,4,5) P3. Using the above-described technique, induction by mutation of PTEN or overexpression of PI (3,4,5) P3 And can be utilized as a therapeutic agent for diseases such as cancer.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

PTEN  Fusion protein production

In the present invention, Ci-VSPTEN21 (Lacroix J et al., J. Biol. Chem. , 286 (20): 17945) obtained from Carlos A. Villalba-Galea (Virginia Commonwealth University, Richmond, Virginia) , Phosphatase domain (PD), C2 domain (C2 domain) excluding the N-terminal phosphopositidylinositol-binding motif (PBM) in the PTEN gene (SEQ ID NO: ; C2) and PDZ binding domain (PDZ-BD) were amplified by PCR. The DNA fragment encodes the 22 to 403th amino acid of the PTEN protein represented by the amino acid sequence of SEQ ID NO: 8.

The amplification was carried out using the following primer set. The primer was prepared by Bioneer Co., Ltd., and the restriction enzyme site was included so that the amplified gene could be inserted into the vector.

[SEQ ID NO: 9]

Forward primer: 5'-AAGCTTCGGACTTAGACTTGACCTATA-3 '

[SEQ ID NO: 10]

Reverse primer: 5'-GGATCCGACTTTTGTAATTTGTGAA-3 '

Then, the 3'-end of CFP-FKBP (coding the amino acid sequence of SEQ ID NO: 12) represented by the nucleotide sequence of SEQ ID NO: 11 and the PTEN DNA fragment indicated by the nucleotide sequence of SEQ ID NO: 13 amplified by the primer set Was inserted into a vector containing the CFP-FKBP gene (CFP-FKBP vector; Takanari Inoue, Johns Hopkins University, School of Medicine).

First, the PTEN DNA fragment of SEQ ID NO: 13 amplified by the primer set was digested with restriction enzymes of HindIII (Enzynomics) and BamHI (Enzynomics), inserted into the CFP-FKBP vector in which the same restriction sites were cut, Finally, a recombinant vector into which a recombinant gene of SEQ ID NO: 4 was inserted was prepared. In addition, the PTEN fusion protein expressed by the recombinant gene was named CF-PTEN.

PI (3,4,5) P3 Dephosphorylation Control ability  Confirm

In the present invention, the PTEN fusion protein prepared in Example 1 is transferred to the cell membrane by an artificial protein transfer technique to selectively phosphorylate PI (3,4,5) P3 into PI (4,5) P2 Respectively.

First, human embryonic kidney 293 cells stably transfected with SV40 T-antigen, obtained from Professor Bertil Hille of the University of Washington School of Medicine, Seattle, Washington. 293tsA1609neo ) TsA201 cells were prepared by culturing in DMEM medium containing 10% FBS and 0.2% penicillin / streptomycin at 5% CO 2 and 37 ° C.

Then, TsA201 cells were cultured in a 35 mm cell culture container for 24 hours. Then, 0.4 μg of CF-PTEN cDNA, 0.2 μg of Btk-PH-GFP and 0.1 μg of LDR (University of Washington, Buffalo Hill) 2000 was treated, transformed for 4 hours, washed and transiently transfected for 24 hours in the same medium as above.

(Btk-PH-GFP; from Carlos A. Villalba-Galea, Virginia Commonwealth University, Richmond, Virginia) of Bruton tyrosine kinase; 5) It was transformed to measure the dephosphorylation of P3 and is known as a probe specific for PI (3,4,5) P3.

The next day, the transformed cells were transferred onto chips coated with poly-L-lysine (0.1 mg / ml, Sigma).

Then, the expression of PTEN protein and the dephosphorylation of PI (3,4,5) P3 were observed using a confocal microscope (treatment with rapamycin (LC Laboratories) at a concentration of 1 μM. Rapamycin was dissolved in DMSO to prepare a stock solution at a concentration of 1 mM. The stock solution was diluted to a concentration of 1 μM in Ringer's solution before use.

Confocal microscopic image observation was performed using a Carl Zeiss LSM 700 confocal microscope (Carl Zeiss AG) at room temperature for 1 to 2 days after the transformation. The Ringer's solution, in which 160 mM NaCl, 2.5 mM KCl, 2 mM CaCl 2 , 1 mM MgCl 2 , 10 mM HEPES, and 8 mM Glucose were added and the pH was adjusted to 7.4 with NaOH, Respectively.

Over time, cell images were scanned at 40X (water) with an objective lens of 512 x 512 pixels using digital zoom. Images were obtained every 5 seconds during this process. Quantitative analysis of the fluorescence intensity in the cytosolic liquid portion of the cytoplasm was performed using the 'measure' tool of ZEN 2012 lite imaging software (Carl Zeiss MicroImaging, Germany).

All confocal images were converted from LSM5 to JPEG format, and raw data over time was processed using Microsoft Office Excel 2012 (Microsoft) and Igor Pro (WaveMetrics, Inc.).

All quantitative data were expressed as mean ± SEM, and Student's T test was used for comparison between the two groups. Comparisons of two or more groups were performed using a one-way ANOVA followed by a Bonferroni post-hoc test. A two-way ANOVA with Bonferroni post-hoc test was used to compare two or more groups that included two independent variables.

As a result, as shown in FIG. 2, it was confirmed that CF-PTEN migrates to an anchored cell membrane by dimerization of FRB and FKBP by addition of rapamycin, and the PD domain of CF-PTEN Specifically, it was confirmed that PI (3,4,5) P3 was dephosphorylated with PI (4,5) P2. This can be confirmed by the movement of Btk-PH-, a biosensor of PI (3,4,5) P3. Also, as shown in the cytosol fluorescence intensity of FIG. 2B, it was confirmed that the addition of rapamycin gradually decreased the anti-CF-PTEN in which the cytosol fluorescence intensity of Btk-PH was rapidly increased.

The fluorescence intensity of the cytosol was decreased by CF-PTEN, which was transferred to the cell membrane by rapamycin treatment. The fluorescence intensity of PI (3,4,5) P3 was dephosphorylated by CF- , 5) the conversion of P2 to Btk-PH-GFP in the cell membrane shifted to the cytosol, indicating increased fluorescence intensity.

Therefore, it has been confirmed that the PTEN fusion protein of the present invention is used as a cell membrane by chemically-inducible dimerization and then selectively dephosphorylated PI (3,4,5) P3. Thus, The protein was confirmed to be applicable to tools for studying intracellular biological phenomena by direct regulation of PI (3,4,5) P3.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments, It will be obvious. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

<110> Daegu Gyeongbuk Institute of Science and Technology <120> PTEN Fusion Protein and Use Thereof <130> 1041877 <160> 13 <170> Kopatentin 2.0 <210> 1 <211> 863 <212> PRT <213> Artificial Sequence <220> <223> CF-PTEN protein amino acid sequence <400> 1 Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val   1 5 10 15 Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu              20 25 30 Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys          35 40 45 Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu      50 55 60 Thr Trp Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln  65 70 75 80 His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg                  85 90 95 Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val             100 105 110 Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile         115 120 125 Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn     130 135 140 Tyr Ile Ser His Asn Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn Gly 145 150 155 160 Ile Lys Ala Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val                 165 170 175 Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro             180 185 190 Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser         195 200 205 Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val     210 215 220 Thr Ala Gly Ily Thr Leu Gly Met Asp Glu Leu Tyr Lys Thr Arg 225 230 235 240 Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu                 245 250 255 Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu             260 265 270 Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln         275 280 285 Lys Asn Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp     290 295 300 Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly 305 310 315 320 Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Tyr Gln Ser                 325 330 335 Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu             340 345 350 Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr         355 360 365 Lys Ser Ala Gly Gly Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp     370 375 380 Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr 385 390 395 400 Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn                 405 410 415 Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp             420 425 430 Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr         435 440 445 Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile     450 455 460 Pro Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu 465 470 475 480 Phe Asp Leu Asp Leu Thr Tyr Ile Tyr Pro Asn Ile Ile Ala Met Gly                 485 490 495 Phe Pro Ala Glu Arg Leu Glu Gly Val Tyr Arg Asn Asn Ile Asp Asp             500 505 510 Val Val Arg Phe Leu Asp Ser Lys His Lys Asn His Tyr Lys Ile Tyr         515 520 525 Asn Leu Cys Ala Glu Arg His Tyr Asp Thr Ala Lys Phe Asn Cys Arg     530 535 540 Val Ala Gln Tyr Pro Phe Glu Asp His Asn Pro Pro Gln Leu Glu Leu 545 550 555 560 Ile Lys Pro Phe Cys Glu Asp Leu Asp Gln Trp Leu Ser Glu Asp Asp                 565 570 575 Asn His Val Ala Ala Ile His Cys Lys Ala Gly Lys Gly Arg Thr Gly             580 585 590 Val Met Ile Cys Ala Tyr Leu Leu His Arg Gly Lys Phe Leu Lys Ala         595 600 605 Gln Glu Ala Leu Asp Phe Tyr Gly Glu Val Arg Thr Arg Asp Lys Lys     610 615 620 Gly Val Thr Ile Pro Ser Gln Arg Arg Tyr Val Tyr Tyr Tyr Ser Tyr 625 630 635 640 Leu Leu Lys Asn His Leu Asp Tyr Arg Pro Val Ala Leu Leu Phe His                 645 650 655 Lys Met Met Phe Glu Thr Ile Pro Met Phe Ser Gly Gly Thr Cys Asn             660 665 670 Pro Gln Phe Val Val Cys Gln Leu Lys Val Lys Ile Tyr Ser Ser Asn         675 680 685 Ser Gly Pro Thr Arg Arg Glu Asp Lys Phe Met Tyr Phe Glu Phe Pro     690 695 700 Gln Pro Leu Pro Val Cys Gly Asp Ile Lys Val Glu Phe Phe His Lys 705 710 715 720 Gln Asn Lys Met Leu Lys Lys Asp Lys Met Phe His Phe Trp Val Asn                 725 730 735 Thr Phe Phe Ile Pro Gly Pro Glu Glu Thr Ser Glu Lys Val Glu Asn             740 745 750 Gly Ser Leu Cys Asp Gln Glu Ile Asp Ser Ile Cys Ser Ile Glu Arg         755 760 765 Ala Asp Asn Asp Lys Glu Tyr Leu Val Leu Thr Leu Thr Lys Asn Asp     770 775 780 Leu Asp Lys Ala Asn Lys Asp Lys Ala Asn Arg Tyr Phe Ser Pro Asn 785 790 795 800 Phe Lys Val Lys Leu Tyr Phe Thr Lys Thr Val Glu Glu Pro Ser Asn                 805 810 815 Pro Glu Ala Ser Ser Thr Ser Val Thr Pro Asp Val Ser Asp Asn             820 825 830 Glu Pro Asp His Tyr Arg Tyr Ser Asp Thr Thr Asp Ser Asp Pro Glu         835 840 845 Asn Glu Pro Phe Asp Glu Asp Gln His Ser Gln Ile Thr Lys Val     850 855 860 <210> 2 <211> 105 <212> PRT <213> Artificial Sequence <220> <223> FKBP protein amino acid sequence <400> 2 Gly Val Gln Val Glu Thr Ile Ser Gly Asp Gly Arg Thr Phe Pro Lys   1 5 10 15 Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu Asp Gly              20 25 30 Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys Met Leu          35 40 45 Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val Ala Gln Met      50 55 60 Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr Ala Tyr  65 70 75 80 Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His Ala Thr Leu Val                  85 90 95 Phe Asp Val Glu Leu Leu Lys Leu Glu             100 105 <210> 3 <211> 403 <212> PRT <213> Artificial Sequence <220> PTEN (PD-C2-PDZ) protein amino acid sequence <400> 3 Met Thr Ala Ile Ile Lys Glu Ile Val Ser Arg Asn Lys Arg Arg Tyr   1 5 10 15 Gln Glu Asp Gly Phe Asp Leu Asp Leu Thr Tyr Ile Tyr Pro Asn Ile              20 25 30 Ile Ala Met Gly Phe Pro Ala Glu Arg Leu Glu Gly Val Tyr Arg Asn          35 40 45 Asn Ile Asp Asp Val Val Arg Phe Leu Asp Ser Lys His Lys Asn His      50 55 60 Tyr Lys Ile Tyr Asn Leu Cys Ala Glu Arg His Tyr Asp Thr Ala Lys  65 70 75 80 Phe Asn Cys Arg Val Ala Gln Tyr Pro Phe Glu Asp His Asn Pro Pro                  85 90 95 Gln Leu Glu Leu Ile Lys Pro Phe Cys Glu Asp Leu Asp Gln Trp Leu             100 105 110 Ser Glu Asp Asp Asn His Val Ala Ala Ile His Cys Lys Ala Gly Lys         115 120 125 Gly Arg Thr Gly Val Met Ile Cys Ala Tyr Leu Leu His Arg Gly Lys     130 135 140 Phe Leu Lys Ala Gln Glu Ala Leu Asp Phe Tyr Gly Glu Val Arg Thr 145 150 155 160 Arg Asp Lys Lys Gly Val Thr Ile Pro Ser Gln Arg Arg Tyr Val Tyr                 165 170 175 Tyr Tyr Ser Tyr Leu Leu Lys Asn His Leu Asp Tyr Arg Pro Val Ala             180 185 190 Leu Leu Phe His Lys Met Met Phe Glu Thr Ile Pro Met Phe Ser Gly         195 200 205 Gly Thr Cys Asn Pro Gln Phe Val Val Cys Gln Leu Lys Val Lys Ile     210 215 220 Tyr Ser Ser Asn Ser Gly Pro Thr Arg Arg Glu Asp Lys Phe Met Tyr 225 230 235 240 Phe Glu Phe Pro Gln Pro Leu Pro Val Cys Gly Asp Ile Lys Val Glu                 245 250 255 Phe Phe His Lys Gln Asn Lys Met Leu Lys Lys Asp Lys Met Phe His             260 265 270 Phe Trp Val Asn Thr Phe Phe Ile Pro Gly Pro Glu Glu Thr Ser Glu         275 280 285 Lys Val Glu Asn Gly Ser Leu Cys Asp Gln Glu Ile Asp Ser Ile Cys     290 295 300 Ser Ile Glu Arg Ala Asp Asn Asp Lys Glu Tyr Leu Val Leu Thr Leu 305 310 315 320 Thr Lys Asn Asp Leu Asp Lys Ala Asn Lys Asp Lys Ala Asn Arg Tyr                 325 330 335 Phe Ser Pro Asn Phe Lys Val Lys Leu Tyr Phe Thr Lys Thr Val Glu             340 345 350 Glu Pro Ser Asn Pro Glu Ala Ser Ser Ser Thr Ser Val Thr Pro Asp         355 360 365 Val Ser Asp Asn Glu Pro Asp His Tyr Arg Tyr Ser Asp Thr Thr Asp     370 375 380 Ser Asp Pro Glu Asn Glu Pro Phe Asp Glu Asp Gln His Ser Gln Ile 385 390 395 400 Thr Lys Val             <210> 4 <211> 2622 <212> DNA <213> Artificial Sequence <220> <223> CF-PTEN protein gene sequence <400> 4 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180 ctcgtgacca ccctgacctg gggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420 aagctggagt acaactacat cagccacaac gtctatatca ccgccgacaa gcagaagaac 480 ggcatcaagg ccaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagacc 720 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc 780 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac 840 aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc 900 cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc 960 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gctaccagtc cgccctgagc 1020 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg 1080 atcactctcg gcatggacga gctgtacaag agtgctggtg gtggagtgca ggtggaaacc 1140 atctccccag gagacgggcg caccttcccc aagcgcggcc agacctgcgt ggtgcactac 1200 accgggatgc ttgaagatgg aaagaaattt gattcctccc gggacagaaa caagcccttt 1260 aagtttatgc taggcaagca ggaggtgatc cgaggctggg aagaaggggt tgcccagatg 1320 agtgtgggtc agagagccaa actgactata tctccagatt atgcctatgg tgccactggg 1380 cacccaggca tcatcccacc acatgccact ctcgtcttcg atgtggagct tctaaaactg 1440 gaatgagctg gtggtagtgc tggtggtcct cgagctcaag cttgacttag acttatccaa 1500 atattattgc tatgggattt cctgcagaaa gacttgaagg tgtatacagg aacaatattg 1560 atgatgtagt aaggtttttg gattcaaagc ataaaaacca ttacaagata tacaatctat 1620 gtgctgagag acattatgac accgccaaat ttaactgcag agttgcacag tatccttttg 1680 aagaccataa cccaccacag ctagaactta tcaaaccctt ctgtgaagat cttgaccaat 1740 ggctaagtga agatgacaat catgttgcag caattcactg taaagctgga aagggacgga 1800 ctggtgtaat gatttgtgca tatttattgc atcggggcaa atttttaaag gcacaagagg 1860 ccctagattt ttatggggaa gtaaggacca gagacaaaaa gggagtcaca attcccagtc 1920 agaggcgcta tgtatattat tatagctacc tgctaaaaaa tcacctggat tacagacccg 1980 tggcactgct gtttcacaag atgatgtttg aaactattcc aatgttcagt ggcggaactt 2040 gcaatcctca gtttgtggtc tgccagctaa aggtgaagat atattcctcc aattcaggac 2100 ccacgcggcg ggaggacaag ttcatgtact ttgagttccc tcagccattg cctgtgtgtg 2160 gtgatatcaa agtagagttc ttccacaaac agaacaagat gctcaaaaag gacaaaatgt 2220 ttcacttttg ggtaaatacg ttcttcatac caggaccaga ggaaacctca gaaaaagtgg 2280 aaaatggaag tctttgtgat caggaaatcg atagcatttg agtatagagc gtgcagataa 2340 tgacaaggag tatcttgtac tcaccctaac aaaaaacgat cttgacaaag caaacaaaga 2400 caaggccaac cgatacttct ctccaaattt taaggtgaaa ctatacttta caaaaacagt 2460 agaggagcca tcaaatccag aggctagcag ttcaacttct gtgactccag atgttagtga 2520 caatgaacct gatcattata gatattctga caccactgac tctgatccag agaatgaacc 2580 ttttgatgaa gatcagcatt cacaaattac aaaagtcgga tc 2622 <210> 5 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> lyn-FRB amino acid sequence <400> 5 Gly Cys Ile Lys Ser Lys Gly Lys Asp Ser Ala Gly Ala Asp Ser Ala   1 5 10 15 Gly Ser Ala Gly Ser Ala Gly Ile Leu Trp His Glu Met Trp His Glu              20 25 30 Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys          35 40 45 Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly      50 55 60 Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp  65 70 75 80 Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn                  85 90 95 Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg             100 105 110 Arg Ile Ser Lys Thr Ser         115 <210> 6 <211> 354 <212> DNA <213> Artificial Sequence <220> <223> lyn-FRB coding gene sequence <400> 6 ggatgtataa aatcaaaagg gaaagacagc gcgggagcag atagtgctgg tagtgctggt 60 agtgctggta tcctctggca tgagatgtgg catgaaggcc tggaagaggc atctcgtttg 120 tactttgggg aaaggaacgt gaaaggcatg tttgaggtgc tggagccctt gcatgctatg 180 atggaacggg gcccccagac tctgaaggaa acatccttta atcaggccta tggtcgagat 240 ttaatggagg cccaagagtg gtgcaggaag tacatgaaat cagggaatgt caaggacctc 300 ctccaagcct gggacctcta ttatcatgtg ttccgacgaa tctcaaagac tagt 354 <210> 7 <211> 1212 <212> DNA <213> Artificial Sequence <220> <223> PTEN gene sequence from VSPEN21 <400> 7 atgacagcca tcatcaaaga gatcgttagc agaaacaaaa ggagatatca agaggatgga 60 ttcgacttag acttgaccta tatttatcca aatattattg ctatgggatt tcctgcagaa 120 agacttgaag gtgtatacag gaacaatatt gatgatgtag taaggttttt ggattcaaag 180 cataaaaacc attacaagat atacaatcta tgtgctgaga gacattatga caccgccaaa 240 tttaactgca gagttgcaca gtatcctttt gaagaccata acccaccaca gctagaactt 300 atcaaaccct tctgtgaaga tcttgaccaa tggctaagtg aagatgacaa tcatgttgca 360 gcaattcact gtaaagctgg aaagggacgg actggtgtaa tgatttgtgc atatttattg 420 catcggggca aatttttaaa ggcacaagag gccctagatt tttatgggga agtaaggacc 480 agagacaaaa agggagtcac aattcccagt cagaggcgct atgtatatta ttatagctac 540 ctgctaaaaa atcacctgga ttacagaccc gtggcactgc tgtttcacaa gatgatgttt 600 gaaactattc caatgttcag tggcggaact tgcaatcctc agtttgtggt ctgccagcta 660 aaggtgaaga tatattcctc caattcagga cccacgcggc gggaggacaa gttcatgtac 720 tttgagttcc ctcagccatt gcctgtgtgt ggtgatatca aagtagagtt cttccacaaa 780 cagaacaaga tgctcaaaaa ggacaaaatg tttcactttt gggtaaatac gttcttcata 840 ccaggaccag aggaaacctc agaaaaagtg gaaaatggaa gtctttgtga tcaggaaatc 900 gatagcattt gcagtataga gcgtgcagat aatgacaagg agtatcttgt actcacccta 960 acaaaaaacg atcttgacaa agcaaacaaa gacaaggcca accgatactt ctctccaaat 1020 tttaaggtga aactatactt tacaaaaaca gtagaggagc catcaaatcc agaggctagc 1080 agttcaactt ctgtgactcc agatgttagt gacaatgaac ctgatcatta tagatattct 1140 gacaccactg actctgatcc agagaatgaa ccttttgatg aagatcagca ttcacaaatt 1200 acaaaagtct ga 1212 <210> 8 <211> 403 <212> PRT <213> Artificial Sequence <220> <223> PTEN amino acid sequence from VSPEN21 <400> 8 Met Thr Ala Ile Ile Lys Glu Ile Val Ser Arg Asn Lys Arg Arg Tyr   1 5 10 15 Gln Glu Asp Gly Phe Asp Leu Asp Leu Thr Tyr Ile Tyr Pro Asn Ile              20 25 30 Ile Ala Met Gly Phe Pro Ala Glu Arg Leu Glu Gly Val Tyr Arg Asn          35 40 45 Asn Ile Asp Asp Val Val Arg Phe Leu Asp Ser Lys His Lys Asn His      50 55 60 Tyr Lys Ile Tyr Asn Leu Cys Ala Glu Arg His Tyr Asp Thr Ala Lys  65 70 75 80 Phe Asn Cys Arg Val Ala Gln Tyr Pro Phe Glu Asp His Asn Pro Pro                  85 90 95 Gln Leu Glu Leu Ile Lys Pro Phe Cys Glu Asp Leu Asp Gln Trp Leu             100 105 110 Ser Glu Asp Asp Asn His Val Ala Ala Ile His Cys Lys Ala Gly Lys         115 120 125 Gly Arg Thr Gly Val Met Ile Cys Ala Tyr Leu Leu His Arg Gly Lys     130 135 140 Phe Leu Lys Ala Gln Glu Ala Leu Asp Phe Tyr Gly Glu Val Arg Thr 145 150 155 160 Arg Asp Lys Lys Gly Val Thr Ile Pro Ser Gln Arg Arg Tyr Val Tyr                 165 170 175 Tyr Tyr Ser Tyr Leu Leu Lys Asn His Leu Asp Tyr Arg Pro Val Ala             180 185 190 Leu Leu Phe His Lys Met Met Phe Glu Thr Ile Pro Met Phe Ser Gly         195 200 205 Gly Thr Cys Asn Pro Gln Phe Val Val Cys Gln Leu Lys Val Lys Ile     210 215 220 Tyr Ser Ser Asn Ser Gly Pro Thr Arg Arg Glu Asp Lys Phe Met Tyr 225 230 235 240 Phe Glu Phe Pro Gln Pro Leu Pro Val Cys Gly Asp Ile Lys Val Glu                 245 250 255 Phe Phe His Lys Gln Asn Lys Met Leu Lys Lys Asp Lys Met Phe His             260 265 270 Phe Trp Val Asn Thr Phe Phe Ile Pro Gly Pro Glu Glu Thr Ser Glu         275 280 285 Lys Val Glu Asn Gly Ser Leu Cys Asp Gln Glu Ile Asp Ser Ile Cys     290 295 300 Ser Ile Glu Arg Ala Asp Asn Asp Lys Glu Tyr Leu Val Leu Thr Leu 305 310 315 320 Thr Lys Asn Asp Leu Asp Lys Ala Asn Lys Asp Lys Ala Asn Arg Tyr                 325 330 335 Phe Ser Pro Asn Phe Lys Val Lys Leu Tyr Phe Thr Lys Thr Val Glu             340 345 350 Glu Pro Ser Asn Pro Glu Ala Ser Ser Ser Thr Ser Val Thr Pro Asp         355 360 365 Val Ser Asp Asn Glu Pro Asp His Tyr Arg Tyr Ser Asp Thr Thr Asp     370 375 380 Ser Asp Pro Glu Asn Glu Pro Phe Asp Glu Asp Gln His Ser Gln Ile 385 390 395 400 Thr Lys Val             <210> 9 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> PTEN forward primer <400> 9 aagcttcgga cttagacttg acctata 27 <210> 10 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> PTEN reverse primer <400> 10 ggatccgact tttgtaattt gtgaa 25 <210> 11 <211> 1440 <212> DNA <213> Artificial Sequence <220> <223> CFP-FKBP coding gene sequence <400> 11 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc 60 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc 120 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc 180 gtgaccaccc tgacctgggg cgtgcagtgc ttcagccgct accccgacca catgaagcag 240 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc 300 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg 360 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag 420 ctggagtaca actacatcag ccacaacgtc tatatcaccg ccgacaagca gaagaacggc 480 atcaaggcca acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac 540 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac 600 ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg 660 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagacccgc 720 gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac 780 ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac 840 gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac 900 aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc 960 gacggccccg tgctgctgcc cgacaaccac tacctgagct accagtccgc cctgagcaaa 1020 gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc 1080 actctcggca tggacgagct gtacaagagt gctggtggtg gagtgcaggt ggaaaccatc 1140 tccccaggag acgggcgcac cttccccaag cgcggccaga cctgcgtggt gcactacacc 1200 gggatgcttg aagatggaaa gaaatttgat tcctcccggg acagaaacaa gccctttaag 1260 tttatgctag gcaagcagga ggtgatccga ggctgggaag aaggggttgc ccagatgagt 1320 gtgggtcaga gagccaaact gactatatct ccagattatg cctatggtgc cactgggcac 1380 ccaggcatca tcccaccaca tgccactctc gtcttcgatg tggagcttct aaaactggaa 1440                                                                         1440 <210> 12 <211> 480 <212> PRT <213> Artificial Sequence <220> <223> CFP-FKBP amino acid sequence <400> 12 Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val   1 5 10 15 Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu              20 25 30 Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys          35 40 45 Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu      50 55 60 Thr Trp Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln  65 70 75 80 His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg                  85 90 95 Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val             100 105 110 Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile         115 120 125 Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn     130 135 140 Tyr Ile Ser His Asn Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn Gly 145 150 155 160 Ile Lys Ala Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val                 165 170 175 Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro             180 185 190 Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser         195 200 205 Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val     210 215 220 Thr Ala Gly Ily Thr Leu Gly Met Asp Glu Leu Tyr Lys Thr Arg 225 230 235 240 Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu                 245 250 255 Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu             260 265 270 Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln         275 280 285 Lys Asn Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp     290 295 300 Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly 305 310 315 320 Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Tyr Gln Ser                 325 330 335 Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu             340 345 350 Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr         355 360 365 Lys Ser Ala Gly Gly Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp     370 375 380 Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr 385 390 395 400 Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn                 405 410 415 Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp             420 425 430 Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr         435 440 445 Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile     450 455 460 Pro Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu 465 470 475 480 <210> 13 <211> 1109 <212> DNA <213> Artificial Sequence <220> PTEN (PD-C2-PDZ) coding gene sequence <400> 13 gacttagact tgacctatat ttatccaaat attattgcta tgggatttcc tgcagaaaga 60 cttgaaggtg tatacaggaa caatattgat gatgtagtaa ggtttttgga ttcaaagcat 120 aaaaaccatt acaagatata caatctatgt gctgagagac attatgacac cgccaaattt 180 aactgcagag ttgcacagta tccttttgaa gaccataacc caccacagct agaacttatc 240 aaacccttct gtgaagatct tgaccaatgg ctaagtgaag atgacaatca tgttgcagca 300 attcactgta aagctggaaa gggacggact ggtgtaatga tttgtgcata tttattgcat 360 cggggcaaat ttttaaaggc acaagaggcc ctagattttt atggggaagt aaggaccaga 420 gacaaaaagg gagtcacaat tcccagtcag aggcgctatg tatattatta tagctacctg 480 ctaaaaaatc acctggatta cagacccgtg gcactgctgt ttcacaagat gatgtttgaa 540 actattccaa tgttcagtgg cggaacttgc aatcctcagt ttgtggtctg ccagctaaag 600 gtgaagatat attcctccaa ttcaggaccc acgcggcggg aggacaagtt catgtacttt 660 gagttccctc agccattgcc tgtgtgtggt gatatcaaag tagagttctt ccacaaacag 720 aacaagatgc tcaaaaagga caaaatgttt cacttttggg taaatacgtt cttcatacca 780 ggaccagagg aaacctcaga aaaagtggaa aatggaagtc tttgtgatca ggaaatcgat 840 agcatttgca gtatagagcg tgcagataat gacaaggagt atcttgtact caccctaaca 900 aaaaacgatc ttgacaaagc aaacaaagac aaggccaacc gatacttctc tccaaatttt 960 aaggtgaaac tatactttac aaaaacagta gaggagccat caaatccaga ggctagcagt 1020 tcaacttctg tgactccaga tgttagtgac aatgaacctg atcattatag atattctgac 1080 accactgact ctgatccaga gaatgaacc 1109

Claims (10)

I) fluorescent protein;
Ii) FKBP (FK506-binding protein) represented by the amino acid sequence of SEQ ID NO: 2; And
Iii) a polypeptide comprising the amino acid sequence of SEQ ID NO: 3 and comprising a phosphatase domain (PD), a C2 domain (C2), a C-terminal PDZ binding domain (PDZ-BD) PTEN protein;
A PTEN fusion protein represented by the amino acid sequence of SEQ ID NO: 1.
The PTEN fusion protein according to claim 1, wherein the fluorescent protein is a green fluorescent protein (GFP), a cyan fluorescent protein (CFP), or a yellow fluorescent protein (YFP) .
delete The method according to claim 1, wherein the PTEN fusion protein has phosphatidylinositol-3,4,5 trisphosphate (PI (3,4,5) P3) Lt; / RTI &gt; fusion protein.
4. A recombinant gene which encodes the PTEN fusion protein of claim 1 and is represented by the nucleotide sequence of SEQ ID NO:
A recombinant vector containing the recombinant gene of claim 5.
delete (a) a) a recombinant vector encoding the PTEN fusion protein of claim 1 or a recombinant vector containing the recombinant gene; And ii) a recombinant vector encoding a Lyn-FRB (FKBP-rapamycin binding domain) fusion protein represented by the amino acid sequence of SEQ ID NO: 5 or a recombinant vector containing the recombinant gene; Expressing the PTEN fusion protein and the Lyn-FRB fusion protein;
(b) treating the cell with rapamycin to transfer the PTEN fusion protein to the cell membrane; And
(c) inducing the dephosphorylation of PI (3,4,5) P3 by the PTEN fusion protein transferred to the cell membrane;
(3, 4, 5) P &lt; 3 &gt;.
[9] The method according to claim 8, wherein the Lyn-FRB fusion protein expressed in step (a) has a Lyn moiety bound to the cell membrane.
[10] The method of claim 8, wherein step (b) is performed using FRB of a Lyn-FRB fusion protein; Rapamycin; A method for regulating the dephosphorylation of PI (3,4,5) P3, wherein the PTEN fusion protein is transferred to the cell membrane by binding of FKBP of the PTEN fusion protein.
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KR20100121710A (en) * 2009-04-30 2010-11-18 한국과학기술원 Method for prevention and treatment of retinal degeneration comprising activity regulation and administration of pten
US20120107299A1 (en) * 2009-04-09 2012-05-03 Miyazaki Prefectural Industrial Support Foundation Phosphorylation-inhibiting agent or dephosphorylating agent for pten

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US20120107299A1 (en) * 2009-04-09 2012-05-03 Miyazaki Prefectural Industrial Support Foundation Phosphorylation-inhibiting agent or dephosphorylating agent for pten
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WO2023172099A1 (en) * 2022-03-11 2023-09-14 재단법인 대구경북과학기술원 Chemogenetically activatable palmitoylation- regulating proteins and uses thereof

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