KR101411296B1 - Liposome transfer signal peptide and method for transferring heterologous protein to fat body using the peptide - Google Patents

Abstract

More particularly, the present invention relates to a gene encoding a signal peptide having an amino acid sequence represented by SEQ ID NO: 1 and an expression comprising a gene encoding a heterologous protein A composition for inducing fat body migration of a heterologous protein comprising the expression vector, and a method for transferring a heterologous protein to an fat body, which comprises introducing the composition into an adipocyte. The signal peptide of the present invention can be fused to a heterologous protein and can be transferred to an adipose tissue of an adipocyte so that the adipocyte is fused to the peptide to transfer the adipose to the adipocyte to selectively and effectively decompose the adipose, And diabetes mellitus-related diseases such as diabetes mellitus can be treated and prevented.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a liposome transfer signal peptide and a method for transferring a heterologous protein to a lipid body using the liposome migration signal peptide,

More particularly, the present invention relates to a gene encoding a signal peptide having an amino acid sequence represented by SEQ ID NO: 1 and an expression comprising a gene encoding a heterologous protein A vector, a composition for inducing migration of a heterologous protein comprising the expression vector in a lipid droplet, and a method for transferring a heterologous protein to an adipocyte, which comprises introducing the composition into an adipocyte.

Fat cells of adipocytes are used for metabolism, and they are responsible for storing and transporting energy as intracellular organelles that store the remaining nutrients in the form of triglycerides. In the case of obesity, the size of the fat body is increased by more than 2 times, and the accumulation of triglyceride is increased. .

The lipid body is surrounded by a single membrane surrounded by triglyceride and cholesterol. Proteins constituting the fat body include adipocyte differentiation-related protein (ADRP), TIP47 and perillipine perilipin). Most of these proteins are expressed specifically in adipocytes, helping to accumulate triglycerides in lipids or protecting lipids from lipolysis by lipase. Therefore, it is possible to remove fat from adipocytes by inhibiting the migration of the protein constituting the fatty body to the fat body or by transferring the lipase to the fat body to induce the degradation of the fat. Currently, the basic mechanism of slimming products is focused on the decomposition of triglycerides of these fatty substances. For example, by treatment with caffeine and the like, hormone sensitive lipase (HSL) Thereby facilitating fat decomposition. However, there is no known signal peptide for inducing migration of the lipolytic enzyme to the fatty body.

On the other hand, the Prp19p protein has the amino acid sequence of SEQ ID NO: 3 and is overexpressed specifically in adipose tissue, and is mainly present in the nucleus and fat body in the differentiated adipocyte. Thus, by suppressing its expression, intracellular lipid formation can be inhibited and the accumulation of triglycerides can be inhibited (PCT Publication No. WO 2006/121250).

Accordingly, the inventors of the present invention continued research to develop a method capable of promoting the degradation of fat by inducing migration of fat-degrading enzymes and the like into fat bodies. As a result, the lipid-derived signal peptide sequence of Prp19p protein was found, The present inventors have completed the present invention by confirming that the heterologous protein can be efficiently transferred to the fat body by using the peptide sequence.

Accordingly, an object of the present invention is to provide an expression vector comprising a gene encoding a signal peptide for transferring a target protein to an adipocyte in a fat cell and a gene encoding the heterologous protein.

It is another object of the present invention to provide a composition for inducing migration of a heterologous protein comprising the expression vector.

It is another object of the present invention to provide a method for transferring a lipolytic enzyme to an fat body using the composition.

According to the above object, the present invention provides a signal peptide having an amino acid sequence represented by SEQ ID NO: 1 and a gene encoding the same.

The present invention also provides an expression vector comprising the gene encoding the signal peptide and the gene encoding the heterologous protein.

In addition, the present invention provides a composition for lipid body migration of a heterologous protein comprising the expression vector.

The present invention also provides a method for transferring a heterologous protein to an oily body using the composition.

Since the signal peptide of the present invention can induce the transfer of the target protein to the fat body in the adipocyte, the lipase is fused with the signal peptide to transfer it to the fat body to promote the fat decomposition, Can be treated and prevented.

The signal peptide of the present invention has the amino acid sequence shown in SEQ ID NO: 1 and corresponds to amino acid sequence number 167-249 including the hydrophobic domain of the Prp19p protein. However, substitution, addition, or deletion of amino acids may be performed within a range that does not affect the function of the protein, and only a part of the protein may be used depending on the purpose. Such modified amino acid sequences are also included within the scope of the present invention. Thus, the present invention also encompasses peptides and fragments thereof having substantially the same sequence as the signal peptide, wherein the substantially identical peptide is at least 80%, preferably at least 90%, most preferably at least 95% ≪ / RTI >

In addition, the present invention provides genes coding for the signal peptide, and these genes have, for example, the gene sequence of SEQ ID NO: 2. However, considering the codon preference codon in the organism to be expressed by the codon degeneracy or the organism to which the gene is expressed, the gene of the present invention may be modified within a range that does not change the amino acid sequence of the protein expressed from the coding region And such modified genes are also included in the scope of the present invention. Accordingly, the present invention also encompasses a gene and a fragment thereof having substantially the same sequence as the gene, wherein substantially the same gene means at least 80%, preferably at least 90%, most preferably at least 95% ≪ / RTI >

The signal peptide and gene of the present invention may be isolated from human tissues or synthesized according to a known gene or peptide synthesis method. For example, the signal peptide of the present invention can be obtained by screening and cloning according to a conventional method based on the gene sequence information of SEQ ID NO: 2. The signal peptide can be produced in large quantities by preparing the expression vector by inserting the produced peptide into a microorganism or an animal cell expression vector known in the art, and introducing it into an appropriate host cell such as E. coli. During the production of the vector, an expression regulatory sequence such as a promoter, a terminator, etc., a self-replicating sequence and the like can be appropriately selected and combined depending on the type of host cell in which the peptide is to be produced.

Also, the present invention provides an expression vector comprising the gene encoding the signal peptide and the gene encoding the heterologous protein. At this time, the heterologous protein fused with the signal peptide is a protein capable of moving into the fat body and exhibiting a significant physiological effect, such as a lipolytic enzyme, protein kinase A (PKA), and a lipolytic enzyme is preferable.

In addition, the present invention provides a composition for inducing migration of lipoproteins of a heterologous protein comprising the expression vector. The composition of the present invention may further comprise a pharmaceutically acceptable carrier, excipient or, if necessary, other additives, in addition to the expression vector used as an active ingredient. Such a composition may be suitably formulated for use as a therapeutic or the like desirable. The composition of the present invention can be administered systemically or locally to a subject requiring decomposition of fat in the fat body, and the dosage can be varied depending on various factors such as the symptom to be treated, the route of administration, the age and weight of the subject, Can be determined.

In addition, the present invention provides a method for transferring a heterologous protein such as a lipolytic enzyme to an fat body, which comprises introducing the composition into an adipocyte. Specifically, common techniques used in the art for intracellular transfection of peptides can be used, for example, by using report transfer lipotransfection and electrophoresis, Move to fat body. When the signal peptide of the present invention is fused with a lipolytic enzyme, the lipolytic enzyme can be easily transferred to an oily body to promote lipolysis without signal transduction by a separate drug treatment such as caffeine. Therefore, the method of inducing fat body movement of lipase by using the signal peptide of the present invention can be used for promoting fat breakdown, which is known as the most common target of obesity treatment, And can be usefully used for the treatment and prevention of abnormal related diseases.

Hereinafter, the present invention will be described in detail with reference to examples.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Example 1: Preparation of several fragments of Prp19p protein and preparation of fusion protein with EGFP

In order to determine which part of the Prp19p protein is involved in the transfer to the fat body, the sequence of the Prp19p protein was determined and the structure was analyzed. As a result, the U-box was found at the N-terminus and the WD repeated at the C- (WD repeat). Further, it was found that a coiled-coil domain forming a dimmer exists between amino acids 72-135 at the N-terminal side, and a hydrophobic domain exists between amino acids 222-228 (Fig. 1 ).

Based on the result of the structural analysis of the Prp19p protein, PCR was carried out using a suitable primer set (Table 1) consisting of the combination of primers 1 to 10 shown in SEQ ID NOS: 4 to 13 and PFU Taq polymerase, To make several Prp19p protein fragments.

The PCR reaction was performed in the following mixture: 38 μl of secondary distilled water, 5 μl of 10 × buffer, 5 μl of dNTP (2.5 mM each), 0.5 μl (100 pmol) of each primer, template (Prp 19p cDNA adipocyte 3T3- L1), and 0.5 μl (5 U / μl) of PFU Taq polymerase.

The PCR was carried out using a Tpersonal, Whatman, GmbH machine and the PCR conditions were as follows: 25 denaturation steps (95 ° C for 30 seconds) after the initial denaturation step (5 min at 95 ° C) (30 min at 55 캜), an extension step (2 min at 68 캜), and a last extension step (at 72 캜 for 10 min). After the PCR reaction, the PCR products were purified using MiniElute ^™ PCR purification kit (QIAGEN, USA).

4 μl of the PCR product was mixed with 1 μl of a salt solution and 1 μl of a pCR 2.1-TOPO vector (Invitrogen) according to the manufacturer's instructions, and reacted at room temperature for 5 minutes. Then, TOP10 E. coli provided in the kit was added. E. coli).

The pCR 2.1-TOPO-Prp19p plasmids obtained above were digested with EcoRI and BamHI restriction enzymes, and the fragments were cloned into the pEGFP-C1 vector, which was cut with EcoRI and BamHI restriction enzymes. Each clone was identified by sequencing.

Example 2: Identification of the signal peptide of Prp19p protein to transfer the external protein to the fat body

<Step 1> Cell differentiation of adipocytes

3T3-L1 (ATCC No. CL-173), an undifferentiated fat cell line of rats, was cultured in DMEM supplemented with 10% bovine serum (Dulbecco's Modified Eagle's Medium, Gibco CA. 1210-0038) And cultured in a 10% CO ₂ incubator until fusion. Subsequently, the cells were incubated with 10% fetal bovine serum, 0.5 mM 3-isobutyl-1-methylxanthine (Sigma), 1 μM dexamethasone (Sigma), and 167 nM insulin (Novo-Nordisk) After culturing for 48 hours in the medium, the medium was replaced with DMEM medium containing 10% fetal bovine serum and 167 nM insulin and cultured again for 48 hours. Finally, differentiated adipocytes were obtained by further culturing in DMEM medium containing only 10% fetal bovine serum for 48 hours.

<Step 2> Expression of fusion proteins of EGFP and Prp19p proteins in adipocytes

The differentiated adipocytes obtained in step 1 were washed twice with PBS, and then 4 g of pEGFP plasmid, pEGFP-Prp19p wild-type plasmid (BD Biosciences, Clontech) or pEGFP-Prp19p fragment plasmid was transfected with Lipofectamine 2000 Invitrogen) for 20 minutes and inserted into the cells. After 8 hours, the cells were washed three times with PBS and further cultured for 48 hours in medium supplemented with 10% fetal bovine serum alone.

<Step 3> Localization of EGFP and Prp19p Fusion Proteins in Adipocytes

Cells obtained in step 2 were washed three times with PBS and fixed with 3% formaldehyde. The cells were then washed three times with PBS containing 0.1% Triton X-100 and then stained with 0.2% Sudan III solution. Cells fixed with 3% formaldehyde were blocked with PBS containing 0.1% Triton X-100 and 10% fetal bovine serum for 20 minutes to stain the original Prp19p protein intracellularly, PRP19 antibody, and anti-rabbit IgG antibody (Amersham) conjugated with FITC (fluorescein isothiocyanate) as a secondary antibody. It was mounted with an anti-fade solution and observed through a microscope.

As a result, when EGFP alone was transfected into fully differentiated adipocytes, the position of the lipid body and EGFP was confirmed. As shown in FIG. 2, the EGFP protein (green) was spread throughout the cell, merged) No yellow color was observed on the image to indicate the same position.

On the other hand, when the intracellular location of the Prp19p protein existing in the original cell and the intracellular location of the Prp19p protein fused with the EGFP were confirmed after transfected into a plasmid in which EGFP-Prp19p was fused, , Prp19p (green) fused to EGFP and Prp19p protein (red) present in the original cell are present around the fat body, and yellow fluorescence is observed when the two fluorescence are present in the same image on the merged image I could confirm.

PCR was carried out using the primers shown in Table 1 with Prp19p cDNA as a template, and DNA fragments in which amino acids were sequentially removed at the N-terminus and C-terminus of the protein, respectively, were obtained. This was ligated to a vector encoding EGFP using EcoRI and BamHI. After plasmids encoding this fusion protein were transfected into adipocytes obtained in step 1, the location of EGFP was observed under a fluorescence microscope.

As a result, as shown in Fig. 4, the Prp19p protein fragment in which the amino acid 1-166 was deleted from the N-terminus was located in the fat body, but in the case of the fragment containing the amino acid 250-504, Since it was lost, the amino acid region 167-249 was found to be important for the transfer to the fatty body. 5, fragments containing amino acids 1-250 and 167-250, respectively, were located in the fat body, but fragments containing amino acids 1-166 and 1-68 were not located in the fat body Therefore, it was confirmed that only the fragments containing the hydrophobic domain consisting of the amino acid sequence of 167-250 have a role of transferring EGFP to fatty substances when they are fused to EGFP.

Example 3: Fat-body transfer peptide of Prp19p protein was used to transfer fat-degrading enzyme to fat body

The signal peptide of SEQ ID NO: 1 and the gene encoding HSL, adipose tissue triglycerol lipase (ATGL) or phospholipase fusion protein as a lipolytic enzyme were amplified by PCR and amplified with pCDNA / HisMAX Invitrogen) to prepare a fusion protein expression vector. This was transfected into adipocytes obtained in step 1 of Example 2 using Lipofectamine 2000 to express the fusion protein by culturing in a medium, and after 48 hours, the medium was collected and the amount of glycerol was measured . Cells expressing the lipase that is not fused with the signal peptide were used as a control, and the amounts of degraded glycerol and the cells expressing the lipase degraded by the peptide were compared.

As a result, in the adipocyte in which the lipase fused to the signal peptide of SEQ ID NO: 1 was expressed, the migration of the lipase into the fat body was accelerated, and the fat was decomposed, thus a larger amount of glycerol was present than the control group Respectively.

Fig. 1 shows the results of structural analysis showing each domain of Prp19p protein,

FIG. 2 shows the results of confirming the position of EGFP in the cells transfected with the plasmid in which only EGFP was fused.

FIG. 3 shows the results of confirming that the intracellular location of the Prp19p protein existing in the original cell and the intracellular position of the Prp19p protein fused with EGFP are identical in the cells transfected with the plasmid in which EGFP-Prp19p is fused,

FIG. 4 shows the result of confirming the intracellular location of the fusion protein by fusing a piece of Prp19p protein cut from N-terminal to EGFP,

FIG. 5 shows the result of confirming the intracellular location of the fusion protein by fusing a piece of Prp19p protein fragments cut from the C-terminal to EGFP.

<110> AMOREPACIFIC CORPORATION <120> SIGNAL PEPTIDE TARGETING TO LIPID DROPLET AND METHOD FOR          TARGETING HETEROLOGOUS PROTEIN TO LIPID DROPLET USING THE SAME <130> FPL / 200707-0203 <160> 13 <170> Kopatentin 1.71 <210> 1 <211> 83 <212> PRT <213> Homo sapiens <400> 1 Val Gly Met Thr Pro Glu Ile Ile Gln Lys Leu Gln Asp Lys Ala Thr   1 5 10 15 Val Leu Thr Thr Glu Arg Lys Lys Arg Gly Lys Thr Val Pro Glu Glu              20 25 30 Leu Val Lys Pro Glu Glu Leu Ser Lys Tyr Arg Gln Val Ala Ser His          35 40 45 Val Gly Leu His Ser Ala Ser Ile Pro Gly Ile Leu Ala Leu Asp Leu      50 55 60 Cys Pro Ser Asp Thr Asn Lys Ile Leu Thr Gly Gly Ala Asp Lys Asn  65 70 75 80 Val Val Val             <210> 2 <211> 249 <212> DNA <213> Homo sapiens <400> 2 ggaatgaccc ctgagattat ccagaagctt caagacaagg ctactgtgct aaccacagag 60 cgtaagaaga gaggaaagac tgtccccgag gagctggtga aacctgaaga gctcagcaag 120 taccggcagg tggcatccca tgtggggcta cacagtgcta gcattcctgg gattctcgct 180 ctggacctgt gtccctcaga caccaacaag attctcactg gtggggcaga taaaaatgtt 240 gttgtcttt 249 <210> 3 <211> 504 <212> PRT <213> Homo sapiens <400> 3 Met Ser Leu Ile Cys Ser Ile Ser Asn Glu Val Pro Glu His Pro Cys   1 5 10 15 Val Ser Pro Val Ser Asn His Val Tyr Glu Arg Arg Leu Ile Glu Lys              20 25 30 Tyr Ile Ala Glu Asn Gly Thr Asp Pro Ile Asn Asn Gln Pro Leu Ser          35 40 45 Glu Gln Leu Ile Asp Ile Lys Val Ala His Pro Ile Arg Pro Lys      50 55 60 Pro Pro Ser Ala Thr Ser Ile Pro Ala Ile Leu Lys Ala Leu Gln Asp  65 70 75 80 Glu Trp Asp Ala Val Met Leu His Ser Phe Thr Leu Arg Gln Gln Leu                  85 90 95 Gln Thr Thr Arg Gln Glu Leu Ser His Ala Leu Tyr Gln His Asp Ala             100 105 110 Ala Cys Arg Val Ile Ala Arg Leu Thr Lys Glu Val Thr Ala Ala Arg         115 120 125 Glu Ala Leu Ala Thr Leu Lys Pro Gln Ala Gly Leu Ile Val Pro Gln     130 135 140 Ala Val Pro Ser Ser Gln Pro Ser Val Val Gly Ala Gly Glu Pro Met 145 150 155 160 Asp Leu Gly Glu Leu Val Gly Met Thr Pro Glu Ile Ile Gln Lys Leu                 165 170 175 Gln Asp Lys Ala Thr Val Leu Thr Thr Glu Arg Lys Lys Arg Gly Lys             180 185 190 Thr Val Pro Glu Glu Leu Val Lys Pro Glu Glu Leu Ser Lys Tyr Arg         195 200 205 Gln Val Ala Ser His Val Gly Leu His Ser Ala Ser Ile Pro Gly Ile     210 215 220 Leu Ala Leu Asp Leu Cys Pro Ser Asp Thr Asn Lys Ile Leu Thr Gly 225 230 235 240 Gly Ala Asp Lys Asn Val Val Val Phe Asp Lys Ser Thr Glu Gln Ile                 245 250 255 Leu Ala Thr Leu Lys Gly His Thr Lys Lys Val Thr Ser Val Val Phe             260 265 270 His Pro Ser Gln Glu Leu Val Phe Ser Ala Ser Pro Asp Ala Thr Ile         275 280 285 Arg Ile Trp Ser Val Pro Asn Thr Ser Cys Val Gln Val Val Arg Ala     290 295 300 His Glu Ser Ala Val Thr Gly Leu Ser Leu His Ala Thr Gly Asp Tyr 305 310 315 320 Leu Leu Ser Ser Asp Asp Gln Tyr Trp Ala Phe Ser Asp Ile Gln                 325 330 335 Thr Gly Arg Val Leu Thr Lys Val Thr Asp Glu Thr Ser Gly Cys Ser             340 345 350 Leu Thr Cys Ala Gln Phe His Pro Asp Gly Leu Ile Phe Gly Thr Gly         355 360 365 Thr Met Asp Ser Gln Ile Lys Ile Trp Asp Leu Lys Glu Arg Thr Asn     370 375 380 Val Ala Asn Phe Pro Gly His Ser Gly Pro Ile Thr Ser Ile Ala Phe 385 390 395 400 Ser Glu Asn Gly Tyr Tyr Leu Ala Thr Ala Ala Asp Ser Ser Ser                 405 410 415 Lys Leu Trp Asp Leu Arg Lys Leu Lys Asn Phe Lys Thr Leu Gln Leu             420 425 430 Asp Asn Phe Glu Val Lys Ser Leu Ile Phe Asp Gln Ser Gly Thr         435 440 445 Tyr Leu Ala Leu Gly Gly Thr Asp Val Gln Ile Tyr Ile Cys Lys Gln     450 455 460 Trp Thr Glu Ile Leu His Phe Thr Glu His Ser Gly Leu Thr Thr Gly 465 470 475 480 Val Ala Phe Gly His His Ala Lys Phe Ile Ala Ser Thr Gly Met Asp                 485 490 495 Arg Ser Leu Lys Phe Tyr Ser Leu             500 <210> 4 <211> 25 <212> DNA <213> Homo sapiens <400> 4 gaattctatg tccctgatct gctcg 25 <210> 5 <211> 25 <212> DNA <213> Homo sapiens <400> 5 gaattctagc atcccagcca ttctg 25 <210> 6 <211> 25 <212> DNA <213> Homo sapiens <400> 6 gaattctgga atgacccctg agatt 25 <210> 7 <211> 25 <212> DNA <213> Homo sapiens <400> 7 gaattctgat aagagtactg agcaa 25 <210> 8 <211> 25 <212> DNA <213> Homo sapiens <400> 8 gaattctggc tgctctctta cctgt 25 <210> 9 <211> 24 <212> DNA <213> Homo sapiens <400> 9 ggatcccaga ctgtagaatt tgag 24 <210> 10 <211> 24 <212> DNA <213> Homo sapiens <400> 10 ggatccgaag gcgatgctgg taat 24 <210> 11 <211> 24 <212> DNA <213> Homo sapiens <400> 11 ggatccaaag acaacaacat tttt 24 <210> 12 <211> 24 <212> DNA <213> Homo sapiens <400> 12 ggatcccacc agctcaccca aatc 24 <210> 13 <211> 24 <212> DNA <213> Homo sapiens <400> 13 ggatccggcg gagggaggct tggg 24

Claims (6)

delete delete 1. A composition for inducing adipose tissue migration of a heterologous protein comprising an expression vector comprising a gene encoding a signal peptide of SEQ ID NO: 1 and a gene encoding a heterologous protein. The method of claim 3, Wherein the heterologous protein is a lipolytic enzyme or protein kinase A (PKA). A method for transferring an in vitro heterologous protein to an fat body, comprising introducing the composition of claim 3 into the adipocyte. 6. The method of claim 5, Wherein said heterologous protein is a lipolytic enzyme or protein kinase A (PKA).

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