KR20080094347A - Surface plasmon resonance imaging detection of specific dna-protein interaction on gold surface - Google Patents
Surface plasmon resonance imaging detection of specific dna-protein interaction on gold surface Download PDFInfo
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Abstract
Description
도 1은 금 표면에 MUA를 붙인 것에, Poly L-lysine을 붙인 것으로써, PDITC(1,4-phenylenediisothiocyanate)를 붙인 것으로써, amine modified DNA를 이중가닥으로 해서 MBP-MITF를 측정한 것이다. FIG. 1 shows MBP-MITF measured by attaching MUA to a gold surface, poly L-lysine, and PDITC (1,4-phenylenediisothiocyanate) to double-stranded amine modified DNA.
도 2은 에폭시(epoxy) 코팅된 유리 슬라이드에 β-CD(β-cyclodextran), MBP(maltose binding protein)-MITF(microphthalmia-associated transcription factor) 융합 단백질, 이중가닥 DNA(E-box)가 차례로 결합하는 단백질 기초 마이크로 칩의 원리를 나타낸 개략도이다. Figure 2 is an epoxy (epoxy) β-CD (β -cyclodextran), the combination then MBP (maltose binding protein) -MITF ( microphthalmia-associated transcription factor) fusion proteins, the double-stranded DNA (E-box) to the coated glass slides It is a schematic diagram showing the principle of a protein-based microchip.
도 3의 A는 단백질 기초 마이크로 칩의 β-CD에 MBP-MITF 융합 단백질을 결합시키기 위하여 제조한 pETMBPek-MITF 발현벡터의 벡터맵이다.Of Figure 3 A is a vector map of a pETMBPek-MITF expression vector prepared in order to combine the MITF-MBP fusion protein to β-CD of the basic protein microchip.
도 3의 B는 상기 벡터를 PCR하여 MITF 유전자 삽입 여부를 확인하고 그 결과를 나타낸 젤(gel) 사진이다. B of FIG. 3 is a gel photograph showing the results of PCR and the MITF gene insertion by the vector.
1: 100bp 래더(ladder);1: 100 bp ladder;
2: 6HUb(Nde Ⅰ/blunt);2: 6HUb ( Nde I / blunt);
3: MITF(blunt/Sal Ⅰ);3: MITF (blunt / Sal I );
4: 3과 동일; 4: same as 3;
5: MITF(EcoR Ⅰ/ Sal Ⅰ); 및5: MITF ( EcoR I / Sal I ); And
6. 5와 동일.6. Same as 5.
도 3의 C는 MBP-MITF 융합 단백질의 친화 컬럼 크로마토그래피(affinity column chromatography) 산물로 SDS-PAGE를 실시한 그림이다. FIG . 3C shows SDS-PAGE of affinity column chromatography products of MBP-MITF fusion proteins.
M: 단백질 마커; M: protein marker;
레인(lane) 1: 전체 분획(total fraction);Lane 1: total fraction;
레인 2: 불용성(insoluble); Lane 2: insoluble;
레인 3: 수용성(soluble);Lane 3: soluble;
레인 4: 로딩 샘플(loading sample);Lane 4: loading sample;
레인 5: 세척 분획(washing fraction); Lane 5: washing fraction;
레인 6-7: 용리 분획(elution fraction); 및 Lanes 6-7: elution fraction; And
레인 8: 한외여과(ultrafiltration) 후의 MBP-MITF. Lane 8: MBP-MITF after ultrafiltration.
도 4의 A는 형광 표지된 MBP-MITF 융합 단백질이 PDITC에 결합하는 반면 대조구인 버퍼(buffer)는 결합하지 않음을 나타낸 SPRI(Surface Plasmon Resonance Imaging) 사진이다. A of Fig. 4 is a SPRI (Surface Plasmon Resonance Imaging) photo showing the other hand, the buffer control (buffer) is not binding fluorescently labeled MBP-fusion protein that binds to the MITF PDITC.
도 4의 B는 SPRI(surface plasmon resonance imaging) 시스템을 이용하여 β-CD와 MBP-MITF는 결합하고 버퍼(buffer)는 결합하지 않음을 확인한 결과를 나타낸 사진이다. B of FIG. 4 The surface plasmon resonance imaging (SPRI) system was used to confirm that β-CD and MBP-MITF bind, but not the buffer (buffer).
도 5는 MBP-MITF 융합 단백질의 농도가 높을수록 MBP-MITF 융합 단백질이 β -CD에 더 많이 결합함을 나타낸 SPRI(surface plasmon resonance imaging) 사진(왼쪽)과 SPRI 분석 그래프(오른쪽)이다. 5 is a surface plasmon resonance imaging (SPRI) photograph (left) and an SPRI analysis graph (right) showing that the higher the concentration of the MBP-MITF fusion protein, the more the MBP-MITF fusion protein binds to β-CD.
도 6은 단백질 기초 마이크로 칩의 MBP-MITF 융합 단백질에 형광 표지된 이중가닥 DNA(E-box), 형광 표지된 돌연변이 이중가닥 DNA, 저해제 + 형광 표지된 이중가닥 DNA(E-box)를 첨가한 후 MBP-MITF와 이중가닥 DNA(E-box)의 결합정도를 나타낸 형광 스캐닝 사진이다. 6 shows the addition of fluorescently labeled double stranded DNA (E-box), fluorescently labeled mutant double stranded DNA, inhibitor + fluorescently labeled double stranded DNA (E-box) to MBP-MITF fusion protein of protein based microchip After the fluorescence scanning picture showing the degree of binding of MBP-MITF and double-stranded DNA (E-box).
도 7의 A는 각각 다른 농도로 MBP-MITF 융합 단백질이 결합된 단백질 기초 마이크로 칩에 형광 표지된 이중가닥 DNA(E-box)를 첨가한 후, MBP-MITF와 이중가닥 DNA(E-box)의 결합정도를 나타낸 형광 스캐닝 사진이다.In Figure 7 A is a basic protein with each of the MBP-MITF fusion protein binding to a different concentration microchip fluorescently labeled double-stranded DNA, MBP-MITF and double-stranded DNA (E-box) was added to (E-box) Fluorescence scanning photograph showing the degree of binding of.
도 7의 B는 각각 다른 농도로 MBP-MITF 융합 단백질이 결합된 단백질 기초 마이크로 칩에 형광 표지된 이중가닥 DNA(E-box)와 형광 표지되지 않은 저해제를 첨가한 후, MBP-MITF와 이중가닥 DNA(E-box)의 결합정도를 나타낸 형광 스캐닝 사진이다. FIG . 7B is a double strand of MBP-MITF and a double strand after adding fluorescently labeled double-stranded DNA (E-box) and an unlabeled inhibitor to the protein-based microchip to which the MBP-MITF fusion protein is bound at different concentrations. Fluorescence scanning photograph showing the degree of binding of DNA (E-box).
도 8의 각각의 색소 색소침착은 MITF와 같은 국산 중국산 약제를 모아 놓은 것으로써, 국산 약제인 독활(Araliae Cordatae Radix)과 중국산인 약제인 영실(Rosae Fructus)과 중국산 약제인 왕불유행(Melandrii Herba)이 있다.Each pigment pigmentation of Figure 8 is a collection of domestic Chinese drugs such as MITF, the domestic drug Araliae Cordatae Radix, the Chinese drug Rosae Fructus and the Chinese drug Melandrii Herba There is this.
본 발명은 MITF 저해제 탐색용 칩 및 이를 이용한 MITF 저해제 탐색방법에 관한 것으로, 구체적으로는 색소 침착과 관련 효소의 유전자 프로머터와 MITF 간의 결합을 억제하여 상기 효소의 발현을 억제하는 MITF 저해제 탐색용 생물학적 마이크로 칩 및 이를 이용한 MITF 저해제 탐색방법에 관한 것이다.The present invention relates to a chip for searching for a MITF inhibitor and a method for searching for a MITF inhibitor using the same, and specifically, to inhibit the expression of the enzyme by inhibiting the pigmentation and binding between the gene promoter of the related enzyme and MITF. The present invention relates to a microchip and a method for searching for MITF inhibitor using the same.
MITF(melanocyte-specific microphthalmia-associated transcription factor isoform)는 멜라닌 세포(melanocyte)에서 특이적으로 발현되는 단백질로 멜라닌(melanin) 생성에 중요한 조절인자이다. 멜라닌은 멜라닌 세포의 골지(Golgi)에서 티로시나제(tyrosinase), Tyrp1(tyrosinase related protein 1), Tyrp2(tyrosinase related protein 2), DCT(dopachrom tautomerase), DHICA(dihyduoxyinkole carboxylic acid) 산화효소 등과 같은 효소에 의해 산화되어 검은색을 내는 유멜라닌(eumelanin)과 황색 또는 적색을 내는 페오멜라닌(pheomelanin)을 생성한다(T. Ellenberger et al . Genes Dev., 8, 970, 1994). 상기 효소들은 특이적으로 멜라닌 세포에서 발현되는데 MITF는 타이로시나제 등 상기 색소형성 효소의 유전자 프로모터에 존재하는 E-box(CATGTG)에 결합하여 E-box에 N-말단 전사 활성 도메인(N-terminal transcription activation domain(TAD)(Sato S et al ., Oncogene 14:3083-3092, 1997), C-말단 TAD(Takeda K et al . Hum Mol Genet 9:125-132, 2000)이 결합하는 것을 촉진함으로써(J. M. Jung et al ., Anal Biochem ., 330, 251, 2004) 색소 침착 효소들의 발현을 유도하여 멜라닌 합성을 야기한다. 따라서 MITF와 E-box 결합은 색소형성에 관여하는 주요 효 소의 발현을 조절하는데 중요한 부분이다. Melanocyte-specific microphthalmia-associated transcription factor isoform (MITF) is a protein that is specifically expressed in melanocytes and is an important regulator of melanin production. Melanin is produced by enzymes such as tyrosinase, tyrosinase related protein 1 (tyrp1), tyrosinase related protein 2 (tyrp2), dopachrom tautomerase (DCT) and dihyduoxyinkole carboxylic acid (DHICA) oxidase in the Golgi of melanocytes. It produces oxidized black eumelanin and yellow or red pheomelanin (T. Ellenberger et. al . Genes Dev ., 8, 970, 1994). The enzymes are specifically expressed in melanocytes. MITF binds to the E-box (CATGTG) present in the gene promoter of the pigment-forming enzyme such as tyrosinase, and the N-terminal transcriptional activation domain (N-) in the E-box. terminal transcription activation domain (TAD) (Sato S et al ., Oncogene 14: 3083-3092, 1997), C-terminal TAD (Takeda K et al . Hum Mol Genet 9: 125-132, 2000) by promoting binding (JM Jung et al ., Anal Biochem . , 330 , 251, 2004) induce the expression of pigmentation enzymes resulting in melanin synthesis. Thus, MITF and E-box binding is an important part of regulating the expression of key enzymes involved in pigmentation.
또한 상기 결합의 저해제로 알려진 MITF-DN, PIAS3(protein inhibitor of activated STAT3)은 관련 효소의 발현을 저해한다고 보고된 바 있어(C. Levy et al. J. Biol . Chem., 277, 1962, 1992) MITF와 E-box의 특이적 결합 저해제는 피부 색소 침착 병변 치료제 및 기능성 화장품의 미백 물질로 사용 가능할 것이라 여겨지고 있다.In addition, MITF-DN and PIAS3 (protein inhibitor of activated STAT3), known as inhibitors of the binding, have been reported to inhibit the expression of related enzymes (C. Levy et. al . J. Biol . Chem ., 277 , 1962, 1992) MITF and E-box specific binding inhibitors are believed to be useful as skin pigmentation lesions and whitening agents for functional cosmetics.
지금까지 이러한 저해제의 탐색방법으로는 gel mobility-shift analysis(C. Jansen et al ., Biochem . J. 246: 227-232, 1987; K. Ruscher et al ., J. Biotechnol . 78: 163-170, 2000), Southwestern blotting(B. Bowen et al ., Nucleic Acids Res . 8: 120, 1980; W.K. Miskimins et al ., Proc . Natl . Acad . Sci. U. S. A. 82: 6741-6744, 1985), ELISA(Y. Choo et al ., Nucleic Acids Res . 21, 1993), reporter constructs in yeast assay(S.D. Hanes et al ., Science 251: 426-430, 1991)가 수행되었으나 상기 방법들은 방사선 동위 원소의 사용, 제한된 실험 수행의 수와 시간, 복잡한 실험 방법 등의 문제점을 가지고 있었다.Until now, the screening method of such inhibitors has been gel mobility-shift analysis (C. Jansen et . al ., Biochem . J. 246: 227-232, 1987; K. Ruscher et al . , J. Biotechnol . 78: 163-170, 2000), Southwestern blotting (B. Bowen et al ., Nucleic Acids Res . 8: 120, 1980; WK Miskimins et al ., Proc . Natl . Acad . Sci. USA 82: 6741-6744, 1985), ELISA (Y. Choo et . al . , Nucleic Acids Res . 21, 1993), reporter constructs in yeast assay (SD Hanes et al . , Science 251: 426-430, 1991), but these methods had problems such as the use of radioisotopes, the limited number and time of conducting experiments, and complex experimental methods.
이에 본 발명자들은 본 발명의 MITF(microphthalmia-associated transcription factor) 저해제 탐색용 단백질 기초 마이크로 칩 및 이를 이용한 MITF 저해제 탐색방법은 말토오즈 결합 단백질(maltose binding protein)과 결합하는 β-CD를 칩 표면에 도입하여 MBP 표지된 단백질만을 특이적으로 결합시키도록 함으로써 기존 DNA 기초 마이크로 칩에서 특이적 결합을 위해 요구되는 바이오 틴(biotin), 알데하이드(aldehyde), 아민(amine) 표지(Jane Linnell et al ., Nucleic Acids Research, 32(4) e44, 2004)와 고순도의 단백질 분리, 정제과정을 생략할 수 있다. 또한 HTS(high throughput screening) 시스템을 적용하여 동시 다발적 실험 수행, 적은 농도 및 작은 분자량의 물질 탐색이 가능하여 미백 관련 표적 단백질인 MITF 저해제를 탐색할 수 있음을 확인함으로써 본 발명을 완성하였다.Therefore, the inventors of the present invention, a protein-based microchip for searching for a microphthalmia-associated transcription factor (MITF) inhibitor of the present invention and a method for searching for a MITF inhibitor using the same, introduce β-CD that binds to a maltose binding protein on the chip surface. Biotin, aldehyde, and amine labels (Jane Linnell et al. ), Which are required for specific binding in existing DNA-based microchips by specifically binding only MBP labeled proteins. al ., Nucleic Acids Research , 32 (4) e44, 2004) and high purity protein separation and purification can be omitted. In addition, by applying a high throughput screening (HTS) system, the present invention was completed by confirming that the MITF inhibitor, a whitening-related target protein, can be searched by performing simultaneous multiple experiments, searching for a small concentration and a small molecular weight material.
본 발명의 목적은 MITF(microphthalmia-associated transcription factor)와 MITF-특이적 DNA를 결합을 저해하는 저해물질 탐색용 칩, 저해물질 탐색용 키트 및 저해물질 탐색방법을 제공하는 것이다.It is an object of the present invention to provide an inhibitor search chip, an inhibitor search kit, and an inhibitor search method that inhibit binding of a microphthalmia-associated transcription factor (MITF) and a MITF-specific DNA.
상기 목적을 달성하기 위하여, 본 발명은 MITF와 MITF-특이적 DNA의 결합을 저해하는 저해물질 탐색용 칩을 제공한다.In order to achieve the above object, the present invention provides a chip for inhibiting the inhibitor to inhibit the binding of MITF and MITF-specific DNA.
또한, 본 발명은 MITF와 MITF-특이적 DNA의 결합을 저해하는 저해물질 탐색용 키트를 제공한다.The present invention also provides a kit for screening inhibitors that inhibits the binding of MITF and MITF-specific DNA.
아울러 본 발명은 MITF와 MITF-특이적 DNA의 결합을 저해하는 저해물질 탐색방법을 제공한다.In addition, the present invention provides a method for screening an inhibitor that inhibits the binding of MITF and MITF-specific DNA.
이하 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명자들은 MIFT와 MITF-특이적 DNA와의 결합을 저해하는 저해자를 탐색하기 위하여 탐색용 마이크로 칩을 제작하였다. 우선 금 기판 위에 MUA, 폴리 L-라이신, PDITC가 순차적으로 결합되어 있으면서, MITF-특이적 DNA와 결합되어 있는 DNA 기초 마이크로 칩을 제조하였으며(도 1 참조), MITF와의 결합이 정상적으로 수행됨을 확인하였다(도 4 참조). 상기 칩는 MITF-특이적 DNA를 고정하여 MITF 및 그의 결합을 저해한다고 추정되는 피검 화합물을 처리하여 MITF와 특이적 DNA와의 결합 저해 여부를 SPRI 방법으로 신속하게 검출할 수 있다. 아울러 본 발명자들은 금 기판에 MUOH, 에피클로로하이드린(epichlorohydrin), 베타 사이클로덱스트린(β-cyclodextrin)이 순차적으로 결합시킨 후 MBP-MITF 융합 단백질을 점적하고, MITF-특이적 DNA를 처리하여 결합 정도를 SPRI 방법으로 확인하였다. 그 결과, 버퍼만을 처리했을 경우와 DNA를 처리했을 경우를 비교했을 확연한 SPRI 결과의 차이를 관찰할 수 있었으며, 아울러 농도의존적으로 그 결합이 증가함을 확인하였다(도 5 참조). 이후 본 발명자들은 DNA를 기초로 한 마이크로 칩에 MITF를 함께 처리하면서 국산 약제인 독활(Araliae Cordatae Radix), 중국산 약제인 영실(Rosae Fructus) 및 중국산 약제 왕불유행(Melandrii Herba)를 함께 농도별로 처리하였고 그중 MITF와 그의 특이적인 DNA의 결합을 저해하는 물질을 탐색하는데 이용하였다(도 8 참조). The inventors have fabricated a microchip for screening to search for inhibitors that inhibit the binding of MIFT to MITF-specific DNA. First, a MUA, a poly L-lysine, and a PDITC were sequentially bound on a gold substrate, and a DNA-based microchip coupled with a MITF-specific DNA was prepared (see FIG. 1), and the binding with the MITF was normally performed. (See Figure 4). The chip can rapidly detect the inhibition of binding of the MITF and specific DNA by treating the test compound that is supposed to inhibit the binding of the MITF and its binding by immobilizing the MITF-specific DNA. In addition, the present inventors sequentially bind MUOH, epichlorohydrin, and beta cyclodextrin to β-cyclodextrin on a gold substrate, and then drop the MBP-MITF fusion protein, and treat the MITF-specific DNA to bind to the degree of binding. Was confirmed by the SPRI method. As a result, a clear difference in SPRI results compared to the case where only the buffer was treated with the case of the DNA treatment was observed, and it was confirmed that the binding increased in a concentration-dependent manner (see FIG. 5). Since the present inventors treated MITF on DNA-based microchips together, we treated the domestic drug Araliae Cordatae Radix, the Chinese drug Rosasil Fructus, and the Chinese drug Melandrii Herba. Among them, it was used to search for substances that inhibit the binding of MITF and its specific DNA (see Fig. 8).
본 발명은 MITF와 MITF-특이적 DNA의 결합을 저해하는 저해물질 탐색용 칩을 제공한다.The present invention provides a chip for screening inhibitors that inhibits the binding of MITF and MITF-specific DNA.
상기 탐색용 칩은 MUOH(11-mercaptoundecanol), 에피클로로하이드린(epichlorohydrin) 및 베타 사이클로덱스트린(β-cyclodextrin)이 수차적으로 결합된 기판 및 상기 베타 사이클로덱스트린에 결합하는 MBP(maltose binding protein)-MITF 융합 단백질로 구성되는 것을 특징으로 한다.The probe chip is a substrate that is aberrantly bonded with MUOH (11-mercaptoundecanol), epichlorohydrin and beta cyclodextrin, and maltose binding protein (MBP) that binds to the beta cyclodextrin. It is characterized by consisting of the MITF fusion protein.
또한, 상기 탐색용 칩은 MUA(11-mercaptoundcanoic acid), 폴리 L-라이신(poly L-lysine) 및 PDITC(1,4-phenylenediisothiocyanate)가 순차적으로 결합된 기판 및 상기 PDITC와 결합하는 MITF-특이적 DNA로 구성되는 것을 특징으로 한다. 상기 DNA는 E-box인 것을 특징으로 하며, 상기 서열은 서열번호 5인 것이 바람직하다. In addition, the search chip is a substrate in which 11-mercaptoundcanoic acid (MUA), poly L-lysine, and PDITC (1,4-phenylenediisothiocyanate) are sequentially bound, and MITF-specific binding to the PDITC. It is characterized by consisting of DNA. The DNA is characterized in that the E-box, the sequence is preferably SEQ ID NO: 5.
또한, 상기 탐색용 칩의 기판은 금(gold)인 것이 바람직하다.In addition, it is preferable that the substrate of the search chip is gold.
또한, 본 발명은 MITF와 MITF-특이적 DNA의 결합을 저해하는 저해물질 탐색용 키트를 제공한다.The present invention also provides a kit for screening inhibitors that inhibits the binding of MITF and MITF-specific DNA.
본 발명의 저해물질 탐색용 키트는 상기 MUOH(11-mercaptoundecanol), 에피클로로하이드린(epichlorohydrin) 및 베타 사이클로덱스트린(β-cyclodextrin)이 수차적으로 결합된 기판 및 상기 베타 사이클로덱스트린에 결합하는 MBP(maltose binding protein)-MITF 융합 단백질로 구성된 탐색용 칩 및 MITF-특이적 DNA를 포함하는 것을 특징으로 한다. 상기 특이적 DNA는 E-box인 것을 특징으로 하며, 상 기 서열은 서열번호 5인 것이 바람직하다. The kit for screening the inhibitor according to the present invention is a substrate in which the MUOH (11-mercaptoundecanol), epichlorohydrin and beta cyclodextrin (beta-cyclodextrin) are aberrantly bound, and MBP (bind to the beta cyclodextrin). maltose binding protein) -MITF fusion protein, characterized in that it comprises a search chip and MITF-specific DNA. The specific DNA is characterized in that the E-box, the sequence is preferably SEQ ID NO: 5.
또한, 본 발명의 저해물질 탐색용 키트는 상기 탐색용 칩은 MUA(11-mercaptoundcanoic acid), 폴리 L-라이신(poly L-lysine) 및 PDITC(1,4-phenylenediisothiocyanate)가 순차적으로 결합된 기판 및 상기 PDITC와 결합하는 MITF-특이적 DNA로 구성된 탐색용 칩 및 MITF 단백질을 포함하는 것을 특징으로 한다. 상기 DNA는 E-box인 것을 특징으로 하며, 상기 서열은 서열번호 5인 것이 바람직하다. In addition, the kit for screening the inhibitor according to the present invention, the detection chip is a substrate in which MUA (11-mercaptoundcanoic acid), poly L- lysine (poly L-lysine) and PDITC (1,4-phenylenediisothiocyanate) are sequentially It is characterized in that it comprises a search chip and MITF protein consisting of MITF-specific DNA binding to the PDITC. The DNA is characterized in that the E-box, the sequence is preferably SEQ ID NO: 5.
아울러 본 발명은 MITF와 MITF-특이적 DNA의 결합을 저해하는 저해물질 탐색방법을 제공한다.In addition, the present invention provides a method for screening an inhibitor that inhibits the binding of MITF and MITF-specific DNA.
본 발명은 하기의 단계로 구성된 방법을 제공한다: 1) MUOH(11-mercaptoundecanol), 에피클로로하이드린(epichlorohydrin) 및 베타 사이클로덱스트린(β-cyclodextrin)이 수차적으로 결합된 기판 및 상기 베타 사이클로덱스트린에 결합하는 MBP(maltose binding protein)-MITF 융합 단백질로 구성된 탐색용 칩에 MITF-특이적 DNA 및 MITF-특이적 DNA와 MITF의 결합을 저해하는 피검 화합물을 첨가하는 단계; 및 2) 단계 1)의 피검 화합물 첨가에 의한 상기 MITF-특이적 DNA와 MITF의 결합저해 정도를 확인하는 단계. The present invention provides a method consisting of the following steps: 1) a substrate in which a number of 11-mercaptoundecanol (MUOH), epichlorohydrin and beta cyclodextrin are aberrationally bound and the beta cyclodextrin Adding a test compound that inhibits the binding of MITF-specific DNA and MITF-specific DNA to MITF to a search chip composed of a maltose binding protein (MBP) -MITF fusion protein that binds to the protein; And 2) confirming the degree of inhibition of binding of the MITF-specific DNA and MITF by adding the test compound of step 1).
또한, 본 발명은 하기의 단계를 구성된 방법을 제공한다: 1) MUA(11-mercaptoundcanoic acid), 폴리 L-라이신(poly L-lysine) 및 PDITC(1,4-phenylenediisothiocyanate)가 순차적으로 결합된 기판 및 상기 PDITC와 결합하는 MITF-특이적 DNA로 구성된 탐색용 칩에 MITF 및 MITF-특이적 DNA와 MITF의 결합을 저해하는 피검 화합물을 첨가하는 단계; 및 2) 단계 1)의 피검 화합물 첨가에 의한 상기 MITF-특이적 DNA와 MITF의 결합저해 정도를 확인하는 단계. The present invention also provides a method comprising the following steps: 1) a substrate in which 11-mercaptoundcanoic acid (MUA), poly L-lysine, and PDITC (1,4-phenylenediisothiocyanate) are sequentially combined; And adding a test compound that inhibits the binding of MITF and MITF-specific DNA and MITF to a search chip composed of MITF-specific DNA binding to the PDITC; And 2) confirming the degree of inhibition of binding of the MITF-specific DNA and MITF by adding the test compound of step 1).
상기 방법들에 있어서, 단계 2)의 결합저해 정도를 확인하는 단계는 SPR(surface plasmon resonance), SPRI(surface plasmon resonance imaging) 및 형광 스캐닝(scanning)으로 구성되는 군으로부터 선택되는 어느 하나의 분석방법을 이용하는 것이 바람직하다.In the above methods, the step of confirming the degree of inhibition of binding of step 2) is any one of analytical methods selected from the group consisting of surface plasmon resonance (SPR), surface plasmon resonance imaging (SPRI) and fluorescence scanning It is preferable to use.
상기 방법들에 있어서, 피검 화합물은 인공 화합물 또는 천연 화합물인 것이 바람직하다.In the above methods, the test compound is preferably an artificial compound or a natural compound.
이하, 본 발명을 실시예에 의해 상세히 설명한다.Hereinafter, the present invention will be described in detail by way of examples.
단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다. However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.
실시예 1> MBPek-MITF 발현벡터 제작Example 1> MBPek-MITF expression vector preparation
<1-1> 시약<1-1> Reagent
3-글리시독실-트리메톡시실란(3-Glycidoxyl-trimethoxysilane), 11-머캅토-1-운데카놀(11-mercapto-1-undecanol), 2-(2-아미노에폭시)-에탄올(2-(2-aminoethoxy)-ethanol), 베타-사이클로덱스트린(beta-cyclodextrin), 에피클로로히드린(epichlorohydrin), 테트라틸렌 글리콜(tetrathylene glycol), 덱스트 란(dextran)은 Sigma 사에서 구입하였으며 소 혈청 알부민(bovine serum albumine)은 Qbiogene 사에서 구입하였다. 미세정렬기(microarrayer)는 프로테오진(CM-100, proteogene)을 사용하였으며 스캐너는 Genfix 4.1 프로그램을 이용하여 확인하였다. SPR은 Autolab 사의 제품을 이용하였으며 SPRI는 한국생명공학연구원 바이오 나노 센터에서 사용하였다.3-glycidoxyl-trimethoxysilane, 11-mercapto-1-undecanol, 2- (2-aminoepoxy) -ethanol (2- (2-aminoethoxy) -ethanol, beta-cyclodextrin, epichlorohydrin, tetratylene glycol and dextran were purchased from Sigma and bovine serum albumin (bovine serum albumine) was purchased from Qbiogene. The microarrayer used proteogene (CM-100, proteogene) and the scanner was identified using Genfix 4.1 program. SPR was used for Autolab's product and SPRI was used for bio nano center of Korea Research Institute of Bioscience and Biotechnology.
<1-2> <1-2> MBPekMBPek -- MITFMITF 발현벡터 제작 Expression vector production
pMAL-c2X 벡터는 New England Biolabs 사에서 구입하였으며 글루타치온-S-트랜스퍼라제(Glutathione-S-transferase, GST)-MITF 벡터는 헤브루 대학 하다싸 메디컬 스쿨 생화학부(department of biochemistry,hebrew university-hadassah,medical school)의 Ehud Razin 박사로부터 수득하였다. 본 실험에서는 상기 pMAL-c2X 벡터를 주형으로 서열번호 1(5’- caa aaa cat atg aaa atc gaa ggt aaa ctg gta - 3’) 및 서열번호 2(5’- gtg cac gaa ttc agt ctg cgc gtc ttt cag ggc tt-3’)로 기재되는 프라이머를 사용하여 하기의 반응조건으로 PCR을 실시하였다. PCR 반응조건은, 표 1과 같이 DNA 중합효소(Unipfu, Takara 사)를 사용하여 상기에 기재된 프라미어 쌍과 주형을 94℃에서 5분 동안 변성시키고, 94℃에서 1분, 55℃에서 1분 및 72℃에서 1분간 30회 반응시키고, 72℃에서 10분간 연장(extension)시켜 반응을 종결하여 MBPek(enterokinase cleavage site)를 수득하였다. MITF도 마찬가지로, 상기 GST-MITF 벡터로부터 MBP와 같은 조건으로 PCR을 실시하여 수득하였다. pET 벡터(Novagen)의 EcoRⅠ, SalⅠ부위에 MBPek와 MITF를 삽입하여 pETMBPek-MITF 발현벡터를 제작하였다(도 3의 A 및 B).The pMAL-c2X vector was purchased from New England Biolabs, and the Glutathione-S-transferase (GST) -MITF vector was derived from the Department of Biochemistry, Hebrew University-hadassah, medical from Dr. Ehud Razin of the school). In this experiment, the pMAL-c2X vector was used as a template for SEQ ID NO: 1 (5'-caa aaa cat atg aaa atc gaa ggt aaa ctg gta-3 ') and SEQ ID NO: 2 (5'- gtg cac gaa ttc agt ctg cgc gtc ttt PCR was performed using the primers described by cag ggc tt-3 ') under the following reaction conditions. PCR reaction conditions, using the DNA polymerase (Unipfu, Takara) as shown in Table 1 to denature the primer pair and template described above for 5 minutes at 94 ℃, 1 minute at 94 ℃, 1 minute at 55 ℃ The reaction was completed 30 times at 72 ° C. for 30 minutes, and the reaction was terminated by extension at 72 ° C. for 10 minutes to obtain MBPek (enterokinase cleavage site). MITF was similarly obtained from the GST-MITF vector by PCR under the same conditions as MBP. MBPek and MITF were inserted into Eco R I and Sal I sites of pET vector (Novagen) to prepare pETMBPek-MITF expression vectors (A and B of FIG. 3).
<실시예 2> MBP-MITF 재조합 융합 단백질 발현 및 분리Example 2 MBP-MITF Recombinant Fusion Protein Expression and Isolation
<2-1> 발현<2-1> expression
pETMBPek-MITF 벡터는 Escherichia coli BL21(DE3)에 형질도입하였다. 흡광도 600nm에서 O.D.가 0.6일 때 최종농도 1 mM로 IPTG(isopropyl-β-D-thiogalactopyranoside)를 처리하여 단백질 발현을 유도하였다. 세포 배양액을 수거하여 6000 rpm에서 10분간 원심분리하여 세포를 수집하고 50 mM Tris-HCl, 10 mM EDTA, pH 8.0의 완충액을 이용하여 세포를 현탁하여 부유시킨 후 초음파를 이용하여 5초 간격으로 5분간 세포 파쇄를 수행하였다. 파쇄된 세포는 50 mM Tris-HCl, 0.5 M NaCl, pH 7.9의 완충용액을 이용하여 수용성 분획을 수집하고 SDS-PAGE로 확인하였다(도 3의 C). pETMBPek-MITF vector was transduced into Escherichia coli BL21 (DE3). When OD was 0.6 at absorbance of 600 nm, protein expression was induced by treatment with IPTG (isopropyl-β-D-thiogalactopyranoside) at a final concentration of 1 mM. Cells were collected and centrifuged at 6000 rpm for 10 minutes to collect cells, suspended and suspended in cells using a buffer of 50 mM Tris-HCl, 10 mM EDTA, pH 8.0. Cell disruption was performed for a minute. The crushed cells were collected by soluble fraction using 50 mM Tris-HCl, 0.5 M NaCl, pH 7.9 buffer and confirmed by SDS-PAGE (FIG. 3C).
<2-2> 분리<2-2> separation
실시예 <2-1>에서 수득한 수용성 분획은 MBP(maltose binding protein) 엑셀로오즈(excellose; Bioprogen)를 충진제로 하여 1.1 cm × 30 cm(Millipore) 컬럼을 이용하였으며 완충액은 20 mM Tris-HCl, 0.2 M NaCl, 1 mM EDTA, pH 7.4로 하고 이에 10 mM 말토오즈를 포함한 것을 용출 완충액으로 이용하여 4 mL/min의 용출속도로 하여 분리하였다. 분리된 단백질은 SDS-PAGE를 이용하여 확인하였다(도 3의 C). The water-soluble fraction obtained in Example <2-1> was used in a 1.1 cm × 30 cm (Millipore) column with MBP (maltose binding protein) Excellose (Bioprogen) as a filler and the buffer solution was 20 mM Tris-HCl. , 0.2 M NaCl, 1 mM EDTA, pH 7.4, and 10 mM maltose was used as the elution buffer and separated at an elution rate of 4 mL / min. The isolated protein was confirmed using SDS-PAGE (FIG. 3C).
<< 실시예Example 3> 3> 이중가닥Double strand DNADNA (E-(E- boxbox )의 제조Manufacturing
이중가닥 올리고뉴클레오타이드(olgonucleotide)는 bHLH-LZ(basic helix-loop-helix-Leucine zipper)서열 중 CATGTG 부분(서열번호 3)을 이용하였으며 Cy3로 표지하였다. 저해제로는 CATGTG 부분을 변형시킨 CTTGAG(서열번호 4, Bioneer 사 제작)를 사용하였다. Cy3의 표지는 Bioneer 사에서 수행하였다(J. M. Jung et al., Anal Biochem ., 330, 251, 2004; M. L. Bulyk et al., Proc . Natl . Acad . Sci ., 98, 7158, 2001) The double stranded oligonucleotide was used as the CATGTG portion (SEQ ID NO: 3) of the basic helix-loop-helix-leucine zipper (bHLH-LZ) sequence and was labeled with Cy3. As an inhibitor, CTTGAG (SEQ ID NO: 4, manufactured by Bioneer) was used. Cy3 labeling was performed by Bioneer (JM Jung et al., Anal Biochem . , 330 , 251, 2004; ML Bulyk et al., Proc . Natl . Acad . Sci ., 98 , 7158, 2001)
<< 실시예Example 4> 유리 및 골드 기판의 수식화( 4> Formulation of glass and gold substrates modificationmodification ))
<4-1> 단백질 기초 마이크로 칩 제작<4-1> Protein-Based Microchip Fabrication
단백질 기초 마이크로 칩 제작을 위하여 골드 기판위에 < 실시예 2>에서 발현 정제한 MBP-MITF 융합 단백질을 고정화하기 위하여 기판 수식화(modification)를 하였다. 먼저 골드표면에 피라나 용액(piranha solution)에 담근 후 60℃에서 15분간 실란화(silanization)를 수행하였고(baylor college of medicine microarray core facility-silanization protocal), 에탄올 및 물로 10회씩 세척하였다. 실란화된 골드 기판은 1 mM 에탄올릭 MUA 용액(ethanolic 11-mercaptoundecanoic acid solution)에 18시간 동안 담구었다가 에탄올 및 물로 10회씩 세척하였다. 이후 N2 가스로 건조하였다. 5mM 에피클로로하이드린은(Epichlorohydrin) 10분동안 담구었다. 이후 버퍼(5 mM boric acid 및 0.1 M NaOH로 pH 8.5로 적정) 1 ml당 0.2 mg β-CD(β-cyclodextrin)가 포함된 액체 용액(aqueous solution; 1 mM per lysine residue)에 담구었다. 이후 N2 가스로 건조하고 물로 헹구어 주었다(도 2). 골드 기판의 제조는 참고문헌의 방법을 이용하여 제조하였다(J. M. Jung et al ., Anal Biochem ., 330, 251, 2004; H. Baek et al., Biotechnol. Biopro. Eng. 9(2): 143-146, 2004).Substrate modification was performed to immobilize the MBP-MITF fusion protein expressed and purified in < Example 2> on a gold substrate for protein-based microchip fabrication. First, soaked in a piranha solution on a gold surface (silanization) was performed for 15 minutes at 60 ℃ (baylor college of medicine microarray core facility-silanization protocal), washed 10 times with ethanol and water. The silanized gold substrate was immersed in 1 mM ethanolic 11-mercaptoundecanoic acid solution for 18 hours and washed 10 times with ethanol and water. Then dried with N 2 gas. 5 mM Epichlorohydrin was soaked for 10 minutes. It was then immersed in an aqueous solution (0.2 mM per lysine residue) containing 0.2 mg β-CD (β-cyclodextrin) per ml of buffer (titrated to pH 8.5 with 5 mM boric acid and 0.1 M NaOH). Then dried with N 2 gas and rinsed with water (Fig. 2). The preparation of the gold substrate was made using the method of reference (JM Jung et. al ., Anal Biochem . , 330 , 251, 2004; H. Baek et al., Biotechnol. Biopro. Eng. 9 (2): 143-146, 2004).
<4-2> DNA 기초 마이크로 칩 제작<4-2> DNA based microchip fabrication
DNA 기초 마이크로 칩 제작을 위하여 골드 기판위에 < 실시예 3>에서 제조한 이중가닥 DNA를 고정화하기 위하여 기판 수식화(modification)를 하였다. 먼저 골드 기판을 피라나 용액(piranha solution)에 담근 후 60℃에서 15분간 실란화(silanization)를 수행하였고(baylor college of medicine microarray core facility-silanization protocal), 에탄올 및 물로 세척을 10회 세척하였다. 실란화된 골드 기판은 1 mM 에탄올릭 MUA 용액(ethanolic 11-mercaptoundecanoic acid solution)에 18시간 동안 담구었다가 물 및 에탄올로 10회 세척하였다. 이후 N2 가스로 건조하였다. 이후 버퍼(5 mM boric acid 및 0.1 M NaOH로 pH 8.5로 적정) 1 ml당 0.2 mg poly L-라이신(lysine)이 포함된 액체 용액(aqueous solution; 1 mM per lysine residue)에 담구었다. 이후 N2 가스로 건조하고 물로 헹구어 주었다. 이후 0.2%(w/v) PDITC(1,4-phenylenediiosithiocyanate)가 포함된 용액(10% pyridine, 90% dimethylformamide)에 실온에서 2시간 동안 골드 기판을 담구고 N2 가스로 건조하였다. 이후 5’a-amine-terminated oligonucleotide(0.5 M NaCl을 포함하는 0.1 M sodium bicarbonateted buffer, pH 9.0에 실시예 3에서 제조된 이중가닥 DNA를 1 mM이 되도록 첨가)와 골드 기판을 37℃에서 2시간 동안 반응시켰다 단백질기기명은 마이크로어레이어고 단백질은 사용 한양은 10㎕그램이다. 2시간 후에 커플 반응(couple reaction)을 중단시키기 위하여, 1% NH4OH의 용액에 실온에서 2분 동안 담구었다. 이후 물로 5분간 행구고 5회 이상 반복적으로 행구어 주었다. 이후 N2 가스로 건조하였다(도 1). 골드 기판의 제조는 참고문헌의 방법을 이용하여 제조하였다(J. M. Jung et al ., Anal Biochem ., 330, 251, 2004; H. Baek et al., Biotechnol. Biopro. Eng. 9(2): 143-146, 2004).Substrate modification was performed to immobilize the double-stranded DNA prepared in < Example 3> on a gold substrate for DNA-based microchip fabrication. First, the gold substrate was immersed in a piranha solution and then silanized for 15 minutes at 60 ° C. (baylor college of medicine microarray core facility-silanization protocal), washed 10 times with ethanol and water. The silanized gold substrate was immersed in 1 mM ethanolic 11-mercaptoundecanoic acid solution for 18 hours and washed 10 times with water and ethanol. Then dried with N 2 gas. It was then immersed in an aqueous solution (1 mM per lysine residue) containing 0.2 mg poly L-lysine (ml) per 1 ml buffer (titrated to pH 8.5 with 5 mM boric acid and 0.1 M NaOH). Then dried with N 2 gas and rinsed with water. Then, the gold substrate was immersed in a solution containing 0.2% (w / v) PDITC (1,4-phenylenediiosithiocyanate) (10% pyridine, 90% dimethylformamide) for 2 hours at room temperature and dried with N 2 gas. Then 5'a-amine-terminated oligonucleotide (0.1 M sodium bicarbonateted buffer containing 0.5 M NaCl, added double-stranded DNA prepared in Example 3 to pH 9.0 to 1 mM) and gold substrate at 37
<< 실시예Example 5> 형광 방법을 이용한 미세정렬( 5> Fine alignment using fluorescence method microarraymicroarray ) 및 데이터 분석) And data analysis
<5-1> 단백질 기초 마이크로 칩의 데이터 분석<5-1> Data Analysis of Protein-based Microchips
< 실시예 2>에서 수득한 MBP-MITF 융합 단백질과 완충액(50 mM Tris, 20%(v/v) 글리세롤 혹은 에틸렌 글리세롤(ethylene glycerol, pH 8.0)을 혼합한 후 스틸스 핀(Stealth pin, Model SMP-10, TeleChem)이 장착된 미세정렬기(Proteogen)를 이용하여 < 실시예 4-1>에서 제작한 기판에 지름 100μm으로 점적하였다(S. H. Baek et al., Biotechnol. Biopro. Eng. 9(2): 143-146, 2004). 농도는 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 및 1 mg/mL로 점적하였으며 마지막으로 1 IgG mg/mL를 점적하였다. 이 때의 습도는 70~80%이며 상온에서 수행되었으며 약 30분간 MBP-MITF를 결합반응시킨 후 세척 완충액(20 mM Tris-buffer, 200 mM NaCl, 1 mM EDTA pH 7.4)과 증류수로 세척하였다. <Example 2> MBP-MITF fusion protein and buffer solution obtained in (50 mM Tris, 20% ( v / v) then a solution of glycerol or ethylene glycerol (ethylene glycerol, pH 8.0) still spin (Stealth pin, Model SMP-10, TeleChem) using a fine aligner (Proteogen) attached <example 4-1> was added dropwise to a diameter of 100μm on the substrate produced in (SH Baek et al., Biotechnol . Biopro. Eng. 9 ( 2): 143-146, 2004) The concentrations were 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1 mg / mL and finally 1 IgG mg / mL. Humidity of 70 ~ 80% was carried out at room temperature and after about 30 minutes combined with MBP-MITF was washed with washing buffer (20 mM Tris-buffer, 200 mM NaCl, 1 mM EDTA pH 7.4) and distilled water.
미세정렬 후 기판 상에 결합된 MBP-MITF에 CoverWell Perfusion Chamber(Sigma)를 이용하여 < 실시예 3>에서 제조한 Cy3 표지 이중가닥 DNA(E-box)와의 결합 반응을 유도하였다. 이를 위하여 상기 coverwell perfusion chamber에 6 mM HEPES, pH 7.8, 40 mM KCl, 0.5 mM EGTG, 6% 글리세롤, 0.25 mg/mL poly-dI-dC, 2% 밀크(milk)와 1 pmol의 Cy3 표지 이중가닥 DNA(E-box)를 혼합하여 처리하였다. 결합시간은 1시간으로 4℃에서 수행하였으며 결합 후 PBS와 PBST(1% Tween-20), PBS/Triton X-100을 이용하여 2번씩 세척하였다. After microalignment, a binding reaction with Cy3-labeled double-stranded DNA (E-box) prepared in < Example 3> was induced using a CoverWell Perfusion Chamber (Sigma) in MBP-MITF bound on the substrate. To this end, 6 mM HEPES, pH 7.8, 40 mM KCl, 0.5 mM EGTG, 6% glycerol, 0.25 mg / mL poly-dI-dC, 2% milk and 1 pmol of Cy3 labeled double strand were added to the coverwell perfusion chamber. DNA (E-box) was mixed and treated. The binding time was performed at 4 ° C. for 1 hour, and then washed twice using PBS, PBST (1% Tween-20) and PBS / Triton X-100.
건조된 칩을 형광 스캐너 Genfix4.1 프로그램을 이용하여 점적된 MBP-MITF에 결합된 형광 이중가닥 DNA(E-box)의 모양과 강도를 확인하였다. 그 결과, MBP-MITF 농도 의존적으로 이중가닥 DNA(E-box)와 결합함을 확인하였다(도 5). The dried chip was checked for the shape and intensity of fluorescent double-stranded DNA (E-box) bound to the MBP-MITF instilled using a fluorescence scanner Genfix4.1 program. As a result, it was confirmed that the binding to double-stranded DNA (E-box) MBP-MITF concentration-dependent (Fig. 5).
<5-2> DNA 기초 마이크로 칩의 데이터 분석<5-2> Data Analysis of DNA-based Microchips
< 실시예 2>에서 수득한 MITF 융합 단백질을 enterokinase로 절단하여 MITF 단백질을 수득하였으며, 완충액(50 mM Tris, 20%(v/v) 글리세롤 혹은 에틸렌 글리세롤(ethylene glycerol, pH 8.0)을 혼합한 후 스틸스 핀(Stealth pin, Model SMP-10, TeleChem)이 장착된 미세정렬기(Proteogen)를 이용하여 < 실시예 4-1>에서 제작한 기판에 지름 100μm으로 점적하였다(S. H. Baek et al., Biotechnol. Biopro. Eng. 9(2): 143-146, 2004). 농도는 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 및 1 mg/mL로 점적하였으며 마지막으로 1mg/ml IGg 를 점적하였다. 이 때의 습도는 70~80%이며 상온에서 수행되었으며 약 30분간 MBP-MITF를 결합반응시킨 후 세척 완충액(20 mM Tris-buffer, 200 mM NaCl, 1 mM EDTA pH 7.4)과 증류수로 세척하였다. The MITF fusion protein obtained in Example 2 was digested with enterokinase to obtain a MITF protein, and then mixed with buffer (50 mM Tris, 20% (v / v) glycerol or ethylene glycerol, pH 8.0). still spin (Stealth pin, Model SMP-10 , TeleChem) using a fine aligner (Proteogen) attached <example 4-1> was added dropwise to a diameter of 100μm on the substrate produced in (SH Baek et al., Biotechnol.Biopro.Eng. 9 (2): 143-146, 2004) Concentrations were instilled at 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1 mg / mL and finally 1 mg / ml. IGg was instilled at a humidity of 70-80% at room temperature, followed by binding reaction with MBP-MITF for about 30 minutes, followed by washing buffer (20 mM Tris-buffer, 200 mM NaCl, 1 mM EDTA pH 7.4). Washed with distilled water.
미세정렬 후 기판 상에 결합된 MBP-MITF에 CoverWell Perfusion Chamber(Sigma)를 이용하여 < 실시예 3>에서 제조한 Cy3 표지 이중가닥 DNA(E-box)와의 결합 반응을 유도하였다. 이를 위하여 상기 coverwell perfusion chamber에 6 mM HEPES, pH 7.8, 40 mM KCl, 0.5 mM EGTG, 6% 글리세롤, 0.25 mg/mL poly-dI-dC, 2% 밀크(milk)와 1 pmol의 Cy3 표지 이중가닥 DNA(E-box)를 혼합하여 처리하였다. After microalignment, a binding reaction with Cy3-labeled double-stranded DNA (E-box) prepared in < Example 3> was induced using a CoverWell Perfusion Chamber (Sigma) in MBP-MITF bound on the substrate. To this end, 6 mM HEPES, pH 7.8, 40 mM KCl, 0.5 mM EGTG, 6% glycerol, 0.25 mg / mL poly-dI-dC, 2% milk and 1 pmol of Cy3 labeled double strand were added to the coverwell perfusion chamber. DNA (E-box) was mixed and treated.
건조된 칩을 형광 스캐너 Genfix4.1 프로그램을 이용하여 점적된 MBP-MITF에 결합된 형광 이중가닥 DNA(E-box)의 모양과 강도를 확인하였다. 그 결과, MBP-MITF 농도 의존적으로 이중가닥 DNA(E-box)와 결합함을 확인하였다(도 4). The dried chip was checked for the shape and intensity of fluorescent double-stranded DNA (E-box) bound to the MBP-MITF instilled using a fluorescence scanner Genfix4.1 program. As a result, it was confirmed that the binding to double-stranded DNA (E-box) MBP-MITF concentration-dependent (Fig. 4).
<< 실시예Example 6> 표면 6> surface 플라즈몬Plasmon 공명( resonance( SurfaceSurface plasmonplasmon resonanceresonance , , SPRSPR ) 데이터 분석Data analysis
<6-1> 단백질 기초 마이크로 칩 분석<6-1> Protein Based Microchip Analysis
SPRI(K-MAC, Manual Type, Korean) 기기에 상기 제조된 골드 기판을 올린 프리즘을 장치하고, 10분 동안 1M NaOH를 흘려주어 안정화시킨 후 완충액(10 mM HEPES, 150 mM NaCl, 0.005% Tween-20)을 흘려주어 세척하였다(S. H. Baek et al., Biotechnol. Biopro. Eng. 9(2): 143-146, 2004). 그 결과, 형광방법을 이용한 실험 결과와 상응하게 SPRI 시스템에서도 MBP-MITF가 이중가닥 DNA(E-box)와 특이적으로 결합함을 확인하였다(도 7). The SPRI (K-MAC, Manual Type, English) device was equipped with a prism with the gold substrate prepared above, stabilized by flowing 1M NaOH for 10 minutes, and then buffered (10 mM HEPES, 150 mM NaCl, 0.005% Tween- 20) was flushed and washed (SH Baek et al., Biotechnol. Biopro. Eng. 9 (2): 143-146, 2004). As a result, it was confirmed that MBP-MITF specifically binds to double-stranded DNA (E-box) in the SPRI system corresponding to the experimental results using the fluorescence method (FIG. 7).
본 SPRI 시스템은 형광방법과는 달리 시료를 Cy3로 표지할 필요가 없고 단백질과 DNA 결합을 실시간으로 분석이 가능하나 동시다발적인 시료 분석이 불가능하다는 단점이 있다. Unlike the fluorescence method, the SPRI system does not need to label the sample with Cy3, and it is possible to analyze protein and DNA binding in real time, but it is impossible to analyze multiple samples simultaneously.
<6-2> DNA 기초 마이크로 칩 분석<6-2> DNA based microchip analysis
SPRI(K-MAC, Manual Type, Korean) 기기에 상기 제조된 골드 기판을 올린 프리즘을 장치하고, (S. H. Baek et al., Biotechnol. Biopro. Eng. 9(2): 143-146, 2004). 각각 MBP-MITF 0.3mg/ml와 IgG 1mg/ml를 1시간 동안 흘려준 후 완충액(10 mM HEPES, 150 mM NaCl, 0.005% Tween-20)을 흘려주어 세척하였다. 1 pmol의 이중가닥 DNA(E-box)를 단백질-DNA 결합 완충용액(6 mM HEPES, pH 7.8, 40 mM KCl, 0.5 mM EGTG, 6% glycerol, 0.25 mg/mL poly-dI-dC, 2% milk)과 혼합하여 1 시간 동안 흘려준 후 완충액(10 mM HEPES, 150 mM NaCl, 0.005% Tween-20)을 흘려주어 세척하여 확인하였다. 그 결과, 형광방법을 이용한 실험 결과와 상응하게 SPRI 시스템에서도 MBP-MITF가 이중가닥 DNA(E-box)와 특이적으로 결합함을 확인하였다(도 4). A SPRI (K-MAC, Manual Type, Korean) device was equipped with a prism loaded with the gold substrate prepared above (S. H. Baek et al., Biotechnol. Biopro. Eng. 9 (2): 143-146, 2004). MBP-MITF 0.3mg / ml and IgG 1mg / ml were flowed for 1 hour, and then washed with a buffer solution (10 mM HEPES, 150 mM NaCl, 0.005% Tween-20). 1 pmol double stranded DNA (E-box) was added to protein-DNA binding buffer (6 mM HEPES, pH 7.8, 40 mM KCl, 0.5 mM EGTG, 6% glycerol, 0.25 mg / mL poly-dI-dC, 2% milk) was mixed for 1 hour and then washed by pouring a buffer solution (10 mM HEPES, 150 mM NaCl, 0.005% Tween-20). As a result, it was confirmed that MBP-MITF specifically binds to double-stranded DNA (E-box) in the SPRI system corresponding to the experimental results using the fluorescence method (FIG. 4).
본 SPRI 시스템은 형광방법과는 달리 시료를 Cy3로 표지할 필요가 없고 단백질과 DNA 결합을 실시간으로 분석이 가능하나 동시다발적인 시료 분석이 불가능하다는 단점이 있다. Unlike the fluorescence method, the SPRI system does not need to label the sample with Cy3, and it is possible to analyze protein and DNA binding in real time, but it is impossible to analyze multiple samples simultaneously.
<< 실시예Example 7> 표면 7> surface 플라즈몬Plasmon 공명 이미지( Resonance image ( SurfaceSurface plasmonplasmon resonanceresonance imageimage , , SPRiSPRi ) 데이터 분석Data analysis
<7-1> 단백질 기초 마이크로 칩 분석<7-1> Protein-Based Microchip Analysis
150-W 석영 텅스텐-할로겐 램프(quartz tungsten-halogen lamp, Schott, Germany), 협대역 간섭 필터(narrow interference filter, 647.1 nm, Δλ=1 nm; Oriel Instruments)가 장착된 SPRI 시스템 내의 프리즘 위에 실험이 완성된 골드 기판을 놓은 후, 1/2 인치 전하 결합 소자(Charge Coupled Device, CCD) 카메라(Sony, Japan)에 의해 반사된 이미지를 확인하였다. 측정 시료 수와 저해제 탐색 방법이 제한적인 SPR의 단점을 보완하고자 SPR에서의 결합을 이미지로 얻을 수 있는 SPRI 방법을 이용하여 확인한 결과, MBP-MITF와 이중가닥 DNA(E-box)가 결합함을 확인하였다. 6 줄 5개씩 점적을 하였으며 농도를 형광 방법과 동일하게 0.06, 0.12, 0.18, 0.24, 0.3 mg/mL로 하여 실험하였다. MBP-MITF의 결합 여부는 0.3mg/ml 이상의 농도에서만 측정이 가능하였고 IgG는 결합하지 못하는 것을 확인하였다. 또한 DNA 크기가 단백질에 비해 매우 작아 결합 여부를 확인하기 불가능하여 DNA를 Cy3로 표지하여 탐지 강도를 높임으로써 농도 의존적인 이중가닥 DNA의 결합을 확인하였다(도 5). 이와 같이 SPRI 시스템은 미세정렬 방법을 이용하여 동시다발적인 시료 분석이 가능하지만 탐지 강도가 낮은 단점이 있었다.Experiments were carried out on a prism in an SPRI system equipped with a 150-W quartz tungsten-halogen lamp (Schott, Germany), a narrow interference filter (647.1 nm, Δλ = 1 nm; Oriel Instruments). After placing the completed gold substrate, the image reflected by the 1/2 inch Charge Coupled Device (CCD) camera (Sony, Japan) was confirmed. In order to make up for the shortcomings of SPR, which are limited in the number of sample and inhibitor detection methods, the SPRI method can be used to obtain the binding of SPR as an image, indicating that MBP-MITF and double-stranded DNA (E-box) bind. Confirmed. Five drops were added in 6 lines, and the concentrations were tested at 0.06, 0.12, 0.18, 0.24, and 0.3 mg / mL in the same manner as the fluorescence method. MBP-MITF binding was measured only at a concentration of 0.3mg / ml or more was confirmed that the IgG can not bind. In addition, the DNA size is very small compared to the protein, it is impossible to determine whether the binding to the DNA by Cy3 to increase the detection intensity confirmed the binding of the concentration-dependent double-stranded DNA (Fig. 5). As such, the SPRI system is capable of simultaneous sample analysis using a microalignment method, but has a disadvantage of low detection intensity.
<7-2> DNA 기초 마이크로 칩 분석 <7-2> DNA based microchip analysis
150-W 석영 텅스텐-할로겐 램프(quartz tungsten-halogen lamp, Schott, Germany), 협대역 간섭 필터(narrow interference filter, 647.1 nm, Δλ=1 nm; Oriel Instruments)가 장착된 SPRI 시스템 내의 프리즘 위에 실험이 완성된 골드 기판을 놓은 후, 1/2 인치 전하 결합 소자(Charge Coupled Device, CCD) 카메라(Sony, Japan)에 의해 반사된 이미지를 확인하였다. 측정 시료 수와 저해제 탐색 방법이 제한적인 SPR의 단점을 보완하고자 SPR에서의 결합을 이미지로 얻을 수 있는 SPRI 방법을 이용하여 확인한 결과, MBP-MITF와 이중가닥 DNA(E-box)가 결합함을 확인하였다. 6 줄 5개씩 점적을 하였으며 농도를 형광 방법과 동일하게 0.06, 0.12, 0.18, 0.24, 0.3 mg/mL로 하여 실험하였다. 6번 줄에 1 mg/ml IgG 을 처리 하여 결합여부를 확인한 결과, MBP-MITF의 결합 여부는 0.3mg/ml 이상의 농도에서만 측정이 가능하였고 IgG는 결합하지 못하는 것을 확인하였다. 또한 DNA 크기가 단백질에 비해 매우 작아 결합 여부를 확인하기 불가능하여 DNA를 Cy3로 표지하여 탐지 강도를 높임으로써 농도 의존적인 이중가닥 DNA의 결합을 확인하였다(도 5). 이와 같이 SPRI 시스템은 미세정렬 방법을 이용하여 동시다발적인 시료 분석이 가능하지만 탐지 강도가 낮은 단점이 있었다.Experiments were carried out on a prism in an SPRI system equipped with a 150-W quartz tungsten-halogen lamp (Schott, Germany), a narrow interference filter (647.1 nm, Δλ = 1 nm; Oriel Instruments). After placing the completed gold substrate, the image reflected by the 1/2 inch Charge Coupled Device (CCD) camera (Sony, Japan) was confirmed. In order to make up for the shortcomings of SPR, which are limited in the number of sample and inhibitor detection methods, the SPRI method can be used to obtain the binding of SPR as an image, indicating that MBP-MITF and double-stranded DNA (E-box) bind. Confirmed. Five drops were added in 6 lines, and the concentrations were tested at 0.06, 0.12, 0.18, 0.24, and 0.3 mg / mL in the same manner as the fluorescence method. As a result of treating 1 mg / ml IgG in
상기와 살펴본 바와 같이, MITF 저해제 탐색용 단백질 기초 마이크로 칩은 기존의 골드기판이 아닌 유리 기판을 이용하여 결제적으로 보급할 수 있으며, 국내에서 주로 개발되는 DNA 기초 마이크로 칩이 아닌 단백질 기초 마이크로 칩으로서 EMSA(eletrophoretic mobility shift assay) 분석에도 이용할 수 있다. 또한 베타 사이클로덱스트란(beta-cycloderxtran)이 MBP(maltose binding protein)의 융합된 단백질과 특이적으로 결합함으로 항체 사용시와 같은 고순도 단백질 정제가 필요치 않고 유리 기판에서 도시 다발적인 실험을 수행할 수 있으면 적은 농도의 저해제 뿐 아니라. 적은 분자량의 저해제 역시 혈광법으로 민감하게 측정하여 스크리닝할 수 있는 장점이 있으므로 MITF 단백질과 이와 결합하는 DNA 사이의 결합을 저해하는 저해제를 탐색 및 발굴하여 화장품 신소재 및 피부질환 치료제를 개발하는데 유 용하게 이용될 수 있다.As described above, the protein-based microchip for searching for MITF inhibitors can be used for payment by using a glass substrate instead of a conventional gold substrate. It can also be used for analysis of eletrophoretic mobility shift assay (EMSA). In addition, the beta-cycloderxtran specifically binds to the fused protein of the maltose binding protein (MBP), which eliminates the need for high-purity protein purification, such as when using antibodies. As well as inhibitors of concentration. Low molecular weight inhibitors also have the advantage of screening sensitively by hemophotometry, which is useful for developing new cosmetics and skin disease treatment agents by searching for and discovering inhibitors that inhibit the binding between MITF proteins and their binding DNA. Can be used.
<110> INHA UNIVERSITY INDUSTRY PARTHNERSHIP INSTITUTE Korea Research Institute of Bioscience and Biotechnology <120> Surface Plasmon Resonance Imaging detection of Specific DNA-protein Interaction on Gold Surface <130> 7p-04-32 <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 33 <212> DNA <213> NMBP-nde I primer <400> 1 caaaaacata tgaaaatcga aggtaaactg gta 33 <210> 2 <211> 35 <212> DNA <213> CMBP-Sma I primer <400> 2 gtgcacgaat tcagtctgcg cgtctttcag ggctt 35 <210> 3 <211> 34 <212> DNA <213> MITF-EcoRI primer <400> 3 ggcgaattca tgctggaaat gctagagtac agtc 34 <210> 4 <211> 32 <212> DNA <213> MITF-Sal I primer <400> 4 ggcgtcgacc taacatgcat gctccgtttc tg 32 <210> 5 <211> 26 <212> DNA <213> E-box <400> 5 ctcacgcccc cggcatgtgg ccgccc 26 <210> 6 <211> 22 <212> DNA <213> competitor <400> 6 ctcacgcccc cggcttgagg cc 22 <110> INHA UNIVERSITY INDUSTRY PARTHNERSHIP INSTITUTE Korea Research Institute of Bioscience and Biotechnology <120> Surface Plasmon Resonance Imaging detection of Specific DNA-protein Interaction on Gold Surface <130> 7p-04-32 <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 33 <212> DNA <213> NMBP-nde I primer <400> 1 caaaaacata tgaaaatcga aggtaaactg gta 33 <210> 2 <211> 35 <212> DNA <213> CMBP-Sma I primer <400> 2 gtgcacgaat tcagtctgcg cgtctttcag ggctt 35 <210> 3 <211> 34 <212> DNA <213> MITF-EcoRI primer <400> 3 ggcgaattca tgctggaaat gctagagtac agtc 34 <210> 4 <211> 32 <212> DNA <213> MITF-Sal I primer <400> 4 ggcgtcgacc taacatgcat gctccgtttc tg 32 <210> 5 <211> 26 <212> DNA <213> E-box <400> 5 ctcacgcccc cggcatgtgg ccgccc 26 <210> 6 <211> 22 <212> DNA <213> competitor <400> 6 ctcacgcccc cggcttgagg cc 22
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