KR100468225B1

KR100468225B1 - Enhanced Inserted YFP

Info

Publication number: KR100468225B1
Application number: KR10-2002-0012409A
Authority: KR
Inventors: 신동승; 박재용; 손호선; 안토니 강; 정연철; 김경진
Original assignee: (주)뉴로제넥스
Priority date: 2002-03-08
Filing date: 2002-03-08
Publication date: 2005-01-26
Also published as: KR20030073037A

Abstract

본 발명은 변종 녹색 형광 단백질 유전자 및 그 발현산물인 형광단백질 (inserted YFP)에 관한 것으로서, 외래의 단백질이나 단백질 부위 (domain)를 코딩하고 있는 염기서열을 삽입하여도 그 발현 산물이 37℃에서 형광을 유지하고 기존의 동종 단백질에 비해 20배에 달하는 형광 강도를 보임을 그 특징으로 하고 있다.The present invention relates to a mutant green fluorescent protein gene and a fluorescent protein (inserted YFP) which is an expression product thereof, and the expression product is fluorescent at 37 ° C. even when a nucleotide sequence encoding a foreign protein or protein domain is inserted. It is characterized by maintaining the fluorescence intensity up to 20 times compared to the existing homologous protein.

본 발명에서는 특정한 아미노산 서열을 형광 단백질 내에 삽입할 수 있도록하기 위하여, DNA 염기서열상에서 특별히 목적한 돌연변이를 도입할 수 있도록 고안된 프라이머를 제조하였고, 이를 이용해서 돌연변이 유발형 PCR (error prone PCR)을 수행하고 이를 포유동물 세포주에서 유전자 운반체로 사용되는 pcDNA3에 클로닝하였다. 제조된 변종 녹색 형광 단백질 유전자를 세포내에 도입하고 그 형광의 강도가 강하게 유지되는 변종 형광단백질을 선별함으로써 여러 염기서열의 삽입이 가능하고, 그 발현산물이 강한 형광을 잃지 않는 변종유전자및 그 변종 녹색 형광 단백질을 발명하게 되었다.In the present invention, in order to be able to insert a specific amino acid sequence in the fluorescent protein, a primer designed to introduce a special target mutation on the DNA sequence was prepared, using the mutagenesis PCR (error prone PCR) And cloned into pcDNA3, which is used as a gene carrier in mammalian cell lines. By introducing the produced mutant green fluorescent protein gene into the cell and selecting the mutant fluorescent protein whose intensity of fluorescence is strongly maintained, the mutant gene and its variant green whose insertion product does not lose strong fluorescence can be inserted. Invented the fluorescent protein.

Description

Inserted green fluorescent protein with enhanced fluorescence intensity and its gene {Enhanced Inserted YFP}

본 발명은 외부의 염기서열을 삽입함으로써 삽입된 염기서열의 세포내에서의 역할을 규명할 수 있도록 사용할 수 있는 형광 단백질에 관한 것이다.The present invention relates to a fluorescent protein that can be used to determine the role of the inserted nucleotide sequence in the cell by inserting an external nucleotide sequence.

녹색 형광 단백질 (GFP)은 유전자의 발현과 그 발현 산물인 단백질의 양적, 위치적 특성을 보고하는 리포터로서 광범위하게 사용되고 있으며 활용분야가 급격히 증가되고 있다. 다양한 목적에 적용되어 오면서 그 특성에 맞도록 고안된 새로운 변종 녹색 형광 단백질들이 만들어져 오고 있었다.Green fluorescent protein (GFP) is widely used as a reporter for reporting the expression of genes and the quantitative and positional properties of the protein, the product of which is expressed, and its applications are rapidly increasing. New varieties of green fluorescent proteins have been created that have been adapted to a variety of purposes and have been adapted to a variety of purposes.

1998년 옥스포드그룹에서 녹색형광단백질 (GFP)내의 여러 위치에 외부 단백질이나 펩타이드를 삽입하여도 그 형광을 유지한다는 보고 (NAR 26:623-630)가 있은 후에, 첸 (R.Tsien) 그룹 등에서 보고한 삽입형광단백질 계열의 분자들이 사용되고 있다. 특히 첸그룹에서 보고하고 있는 삽입형광단백질의 경우 와이에프피인스 (YFPins)와 여기에 칼모듈린이 삽입된 캄가루 (Camgaroo) 등이 있다. 기존의 삽입가능한 녹색 형광 단백질의 경우 1999년에 첸 등이 발표한 논문에 따르면 145번 아미노산인 티로신 (Tyrosine)이 GGTGEL이라는 아미노산 서열로 변경되었고, 이부분에 도입된 KpnI,SacI 제한효소를 이용함으로써 외래의 염기서열을 클로닝 하도록 하였다 (PNAS 96:11241-11246). 이들에 의해서 만들어진 변종 형광 단백질은 37℃에서 형광을 거의 보이지 못하고 있었고, 28℃에서 형광을 보이는 특성을 보이고 있었다. 때문에 포유동물 세포주에서의 여러가지 세포내 활성을 측정하는 데에는 사용할 수 없다는 단점을 보이고 있었다. 같은 그룹에 따르면 이 캄가루 (Camgaroo)를 돌연변이를 유발시켜 얻은 69번 아미노산인 글루타민이 메치오닌으로 변한 Q69M돌연변이는 37℃에서 형광을 보이고 있는 것으로 보고되었다 (JBC,276:29188~29194).In 1998, the Oxford Group reported that fluorescence was maintained even when foreign proteins or peptides were inserted at various positions in the green fluorescent protein (GFP) (NAR 26: 623-630), and then reported by the R.Tsien group and others. An insertional fluorescent protein family of molecules is in use. In particular, YFPins, which are reported by the Chen Group, and Camgaroo, which contain calmodulin, are also included. In the case of the conventional insertable green fluorescent protein, a paper published by Chen et al. In 1999 showed that the 145 amino acid tyrosine (Tyrosine) was changed to an amino acid sequence called GGTGEL and by using the KpnI and SacI restriction enzymes introduced therein. The foreign sequence was cloned (PNAS 96: 11241-11246). The mutant fluorescent proteins made by them showed little fluorescence at 37 ° C and showed fluorescence at 28 ° C. Therefore, it has been shown that it cannot be used to measure various intracellular activities in mammalian cell lines. According to the same group, the Q69M mutation, in which glutamine, amino acid 69 obtained by mutating Camgaroo, was converted to methionine, was reported to fluoresce at 37 ° C (JBC, 276: 29188-29194).

본 발명자 등이 동일한 유전자를 제조하여 포유동물세포에 도입한 후 공초점 현미경으로 상을 잡아 본 결과 그 형광 강도가 매우 미약하여서 단백질의 결합 부위를 삽입하여 그 결합성의 변화를 보고자하는 연구나, 단백질 분해 효소에 대한 절단 부위를 삽입하는 등의 활용,그리고 세포내의 칼슘 농도 측정 등이 거의 불가능함을 알 수 있었다. 따라서 외래 유전자의 삽입을 통한 연구가 현실적으로 가능하려면 삽입형광단백질의 형광 강도가 현저히 증가된 변종을 선별하고 이에 관한 연구가 선행되어야 했다.The present inventors prepared the same gene and introduced it into mammalian cells, followed by confocal microscopy, and found that the fluorescence intensity was very weak. Application of the cleavage site for the degrading enzyme and measurement of intracellular calcium concentration were almost impossible. Therefore, in order to be able to study through the insertion of a foreign gene in reality, it was necessary to select a strain that significantly increased the fluorescence intensity of the inserted fluorescent protein and to study it.

본 발명은 이에 관한 것으로서 기존의 변종 단백질에서 제외되었던 145번 아미노산인 티로신을 도입하고 pcDNA3 운반체에 효율적으로 클로닝할 수 있도록 두 개의 제한효소 인지 부위 (BamHI, 및 NheI )를 도입하고 동시에 적절한 펩타이드 연결 부위를 도입할 수 있도록 프라이머를 디자인 하였고, 이를 사용하여 돌연변이를 많이 유발할 수 있도록 하는 피씨알 클로닝을 수행함으로써 빛이 강하게 유지되는 변종 형광 단백질을 개발하고자 하였다.The present invention relates to the introduction of two restriction enzyme recognition sites (BamHI, and NheI) at the same time to introduce the tyrosine amino acid 145, which has been excluded from the existing variant proteins and to efficiently clone the pcDNA3 carrier, and at the same time an appropriate peptide linking site The primers were designed to introduce and used to develop a fluorescence protein that maintains light strongly by performing PCR cloning to induce a lot of mutations.

이 방법으로 선별된 변종 형광 단백질은 145번 아미노산 부위에 YGGSGAS라는 아미노산 서열을 갖는 특징이 있다. 이러한 특징을 갖는 변종 형광 단백질을 와이시트린 (Y-Citrine)이라 명명하였다. 이 부위는 기존의 변종 형광 단백질의 펩타이드 연결 부위와 비교하여 전기적성질을 거의 띠지 않고, 연결부위로서 작용할 수 있도록 고안되었고, 염기서열 상에서 유전자 운반체에는 존재하지 않는 제한 효소 인지 부위를 가지게 됨으로써 사용하는 유전자 운반체에서의 클로닝작업을 일회로 마칠 수 있는 특성을 가지게 된다.The variant fluorescent protein selected by this method is characterized by having an amino acid sequence of YGGSGAS at amino acid site 145. The variant fluorescent protein with this feature was named Y-Citrine. This site is designed to act as a linking site with little electrical properties compared to the peptide linking site of the conventional fluorescence fluorescent protein, and has a restriction enzyme recognition site that does not exist in the gene carrier on the nucleotide sequence. It has the characteristics to complete the cloning operation in the carrier.

또한 이 과정에서 새로운 돌연변이가 유발된 변종을 선별하였는데 이 변종단백질은 와이시트린이 가지고 있는 돌연변이 외에 192번 아미노산이 프롤린에서 라이신 (P192L)으로 변이 된것을 확인하였고 이를 페리도트 (Peridot)이라 명명하였다. 이는 와이시트린과 더불어 또 다른 삽입형 형광 단백질로서의 활용을 가능하게 해주는 것이었다.In the process, a new mutation-induced strain was selected. In addition to the mutations of Y-citrine, the mutant protein was found to have amino acid 192 mutated from proline to lysine (P192L), which was named Peridot. This, in combination with ycitrine, enabled the use as another implantable fluorescent protein.

본 발명에서 보여주고 있는 이 두종의 형광 단백질은 기존의 변종 형광 단백질에 비해 공초점 현미경에서 약 20배의 형광 강도를 보이고 있는 것으로 측정되었고, 이는 세포 수준의 약효평가에 사용할 수 있는 새로운 리포터의 출현을 의미하는 것이라 할 수 있다. 이렇게 새로이 만들어진 리포터 단백질을 이용하여 그 삽입부위에 단백질 분해 효소의 절단 부위, 단백질 결합부위등을 삽입하고 그 산물의 형광을 분석함으로써 세포내에서 벌어지는 다양한 생명현상을 분석할 수 있는 길을 열어 놓은 것이라 할 수 있다.The two fluorescent proteins shown in the present invention were measured to show about 20 times the fluorescence intensity under confocal microscopy compared to the existing mutant fluorescent proteins, which resulted in the emergence of a new reporter that can be used for cell-level drug efficacy evaluation. It can be said to mean. Using the newly produced reporter protein, the cleavage site and protein binding site of protease were inserted into the insertion site, and the fluorescence of the product was analyzed to open a way to analyze various life phenomena occurring in cells. can do.

본 발명에서 개발하고자 한 것은 형광의 강도가 증강된 삽입형 황색 편이 녹색 형광 단백질 (inserted YFP)의 개발이었다. 이를 위해서는 새로운 높은 형광 강도를 보이는 돌연변이의 선별, 펩타이드 연결부위의 엔지니어링, 간편한 클로닝을 위한 제한효소 인지부위의 도입등이 필요하였다.What was intended to be developed in the present invention was the development of an inserted yellow shift green fluorescent protein (inserted YFP) with enhanced fluorescence intensity. This required the selection of new high fluorescence intensity mutations, engineering of peptide linkages and introduction of restriction enzyme recognition sites for easy cloning.

도 1a는 헬라(HeLa) 세포에 제조된 삽입형광단백질을 도입한 후 공초점 현미경으로 촬영한 A) Citrine-Ins, B) Y-Citrine, C) Peridot, D) Y-Citrine-5AB, E) Peridot-5AB의 형광사진Figure 1a is a) Citrine-Ins, B) Y-Citrine, C) Peridot, D) Y-Citrine-5AB, E) taken by confocal microscopy after introducing the inserted fluorescent protein prepared in HeLa cells Fluorescence of Peridot-5AB

도 1b는 도 1a의 이미지를 정량화하여 제조된 삽입형광단백질들의 형광 강도를 비교한 그림Figure 1b is a comparison of the fluorescence intensity of the fluorescent proteins prepared by quantifying the image of Figure 1a

본 발명은 간편한 클로닝을 위해 제한 효소 인지 서열 (BamHI 및 NheI )를 도입하였다. 이는 현재 사용하고 있는 포유동물 세포용 유전자 운반체인 pcDNA3에는 존재하지 않는 제한 효소 인지부위이다. 이 제한 효소 인지 부위를 포함하는 YGGSGAS의 펩타이드 연결부위를 삽입하였다. 이 펩타이드 연결부위는 기존의 GGTGEL에 비해 전기적 성질을 최소화 하였고 이로 인한 구조적 변화를 줄이고자 하였다.The present invention introduced restriction enzyme recognition sequences (BamHI and NheI) for convenient cloning. This is a restriction enzyme recognition site that does not exist in pcDNA3, which is a gene carrier for mammalian cells currently in use. The peptide linkage region of YGGSGAS containing this restriction enzyme recognition site was inserted. The peptide linkages have minimized the electrical properties compared to the existing GGTGEL and attempted to reduce the structural changes.

또한 이 변이와 PCR 과정을 통해 도입된 새로운 돌연변이에 의해 강한 형광을 보이는 변종 형광 단백질을 포유동물 세포주인 헬라 (HeLa) 세포주에서 선별함으로써 발명을 완성할 수 있었다. 도 1a에 나타낸 바와 같이 새로이 제조된 삽입형광단백질은 기존의 첸 그룹의 삽입형광단백질인 시트린인스 (Citrine-ins)에 비해 현저히 증강된 형광 강도를 보임을 이미지를 통해 알 수 있었고, 도1b에 나타낸 바와 같이 이를 공초점 현미경 하에서 정량한 결과 20배 정도의 형광 강도를 보이는 것을 알 수 있었다.In addition, the invention could be completed by selecting a variant fluorescent protein showing strong fluorescence by the mutation and a new mutation introduced through the PCR process in the HeLa cell line, a mammalian cell line. As shown in FIG. 1A, the newly prepared insert fluorescent protein showed significantly increased fluorescence intensity compared to the existing Chen group of insert fluorescent protein Citrine-ins, and the image was shown in FIG. 1B. As a result of quantification under confocal microscope, the fluorescence intensity was about 20 times.

이들 선별된 변종 형광 단백질 와이씨트린과 페리도트의 삽입부위에 사람의 C형 간염 바이러스의 단백질 분해효소 (protease)인 NS3 단백질에 의한 절단 부위의 하나인 5AB 연결부위의 염기서열을 삽입하였을 때에도 그 형광을 유지하였고 이를 통해 세포수준에서의 C형 간염 바이러스의 단백질 분해효소의 활성을 평가할 수 있는 방법의 개발 등을 가능하게 하였다.When the nucleotide sequence of the 5AB linking site, which is one of the cleavage sites by the NS3 protein, which is a protease of the hepatitis C virus, is inserted into the insertion sites of the selected fluorescence protein Ycytrin and peridot In this way, it was possible to develop a method for evaluating the protease activity of hepatitis C virus at the cellular level.

본 발명은 포유동물 세포내에서 (37℃) 기존의 동종 단백질에 비해 그 형광강도가 20배에 달하는 삽입형광단백질을 개발함으로써 다양한 단백질 부위의 삽입을 통한 연구를 현실화 할 수 있었다는 것과 포유동물 세포형 유전자 운반체에서의 클로닝이 한 단계로 간편해 졌다는 것을 특징으로 한다.According to the present invention, the development of an insert fluorescent protein having a fluorescence intensity of 20 times higher than that of a conventional homologous protein in a mammalian cell (37 ° C) has made it possible to realize research through the insertion of various protein sites and mammalian cell types Cloning in gene carriers has been simplified in one step.

제조된 변종 형광단백질의 삽입부위에 C형 간염바이러스의 NS3 단백질 분해효소(protease)의 절단부위인 5AB를 삽입하였을 때에도 그 형광을 유지한다는 데에서 5AB삽입형광단백질(Y-Citrine-5AB 및 Peridot-5AB)를 기질로 이용하는 C형간염 바이러스 단백질 분해효소 활성 분석 시스템(HCV protease assay system)의 개발이 가능해졌음을 알 수 있다.When 5AB, a cleavage site of NS3 protease of hepatitis C virus, was inserted into the inserted fluorescent protein, the 5AB insertion fluorescent protein (Y-Citrine-5AB and Peridot- was maintained. It can be seen that the development of a hepatitis C virus protease assay system using 5AB) as a substrate has been made possible.

<110> NEUROGENEX Co.Ltd. <120> Enhanced inserted YFP <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 738 <212> DNA <213> Aequorea victoria <220> <221> CDS <222> (1)..(735) <223> y-citrine DNA sequence <400> 1 atg gtg agc aag ggc gag gag ctg ttc acc ggg gtg gtg ccc atc ctg 48 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 1 5 10 15 gtc gag ctg gac ggc gac gta aac ggc cac aag ttc agc gtg tcc ggc 96 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 20 25 30 gag ggc gag ggc gat gcc acc tac ggc aag ctg acc ctg aag ttc atc 144 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 35 40 45 tgc acc acc ggc aag ctg ccc gtg ccc tgg ccc acc ctc gtg act acc 192 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50 55 60 ttc ggc tac ggc ctg atg tgc ttc gcc cgc tac ccc gac cac atg aag 240 Phe Gly Tyr Gly Leu Met Cys Phe Ala Arg Tyr Pro Asp His Met Lys 65 70 75 80 cag cac gac ttc ttc aag tcc gcc atg ccc gaa ggc tac gtc cag gag 288 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 85 90 95 cgc acc atc ttc ttc aag gac gac ggc aac tac aag acc cgc gcc gag 336 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100 105 110 gtg aag ttc gag ggc gac acc ctg gtg aac cgc atc gag ctg aag ggc 384 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125 atc gac ttc aag gag gac ggc aac atc ctg ggg cac aag ctg gag tac 432 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135 140 aac tac ggt gga tcc ggt gct agc aac agc cac aac gtc tat atc atg 480 Asn Tyr Gly Gly Ser Gly Ala Ser Asn Ser His Asn Val Tyr Ile Met 145 150 155 160 gcc gac aag cag aag aac ggc atc aag gtg aac ttc aag atc cgc cac 528 Ala Asp Lys Gln Lys Asn Gly Ile Lys Val Asn Phe Lys Ile Arg His 165 170 175 aac atc gag gac ggc agc gtg cag ctc gcc gac cac tac cag cag aac 576 Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn 180 185 190 acc ccc atc ggc gac ggc ccc gtg ctg ctg ccc gac aac cac tac ctg 624 Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu 195 200 205 agc tac cag tcc gcc ctg agc aaa gac ccc aac gag aag cgc gat cac 672 Ser Tyr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His 210 215 220 atg gtc ctg ctg gag ttc gtg acc gcc gcc ggg atc act ctc ggc atg 720 Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met 225 230 235 240 gac gag ctg tac aag taa 738 Asp Glu Leu Tyr Lys 245 <210> 2 <211> 245 <212> PRT <213> Aequorea victoria <400> 2 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 1 5 10 15 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 20 25 30 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 35 40 45 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50 55 60 Phe Gly Tyr Gly Leu Met Cys Phe Ala Arg Tyr Pro Asp His Met Lys 65 70 75 80 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 85 90 95 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100 105 110 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135 140 Asn Tyr Gly Gly Ser Gly Ala Ser Asn Ser His Asn Val Tyr Ile Met 145 150 155 160 Ala Asp Lys Gln Lys Asn Gly Ile Lys Val Asn Phe Lys Ile Arg His 165 170 175 Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn 180 185 190 Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu 195 200 205 Ser Tyr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His 210 215 220 Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met 225 230 235 240 Asp Glu Leu Tyr Lys 245 <210> 3 <211> 738 <212> DNA <213> Aequorea victoria <220> <221> CDS <222> (1)..(735) <223> Peridot DNA sequence <400> 3 atg gtg agc aag ggc gag gag ctg ttc acc ggg gtg gtg ccc atc ctg 48 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 1 5 10 15 gtc gag ctg gac ggc gac gta aac ggc cac aag ttc agc gtg tcc ggc 96 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 20 25 30 gag ggc gag ggc gat gcc acc tac ggc aag ctg acc ctg aag ttc atc 144 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 35 40 45 tgc acc acc ggc aag ctg ccc gtg ccc tgg ccc acc ctc gtg act acc 192 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50 55 60 ttc ggc tac ggc ctg atg tgc ttc gcc cgc tac ccc gac cac atg aag 240 Phe Gly Tyr Gly Leu Met Cys Phe Ala Arg Tyr Pro Asp His Met Lys 65 70 75 80 cag cac gac ttc ttc aag tcc gcc atg ccc gaa ggc tac gtc cag gag 288 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 85 90 95 cgc acc atc ttc ttc aag gac gac ggc aac tac aag acc cgc gcc gag 336 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100 105 110 gtg aag ttc gag ggc gac acc ctg gtg aac cgc atc gag ctg aag ggc 384 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125 atc gac ttc aag gag gac ggc aac atc ctg ggg cac aag ctg gag tac 432 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135 140 aac tac ggt gga tcc ggt gct agc aac agc cac aac gtc tat atc atg 480 Asn Tyr Gly Gly Ser Gly Ala Ser Asn Ser His Asn Val Tyr Ile Met 145 150 155 160 gcc gac aag cag aag aac ggc atc aag gtg aac ttc aag atc cgc cac 528 Ala Asp Lys Gln Lys Asn Gly Ile Lys Val Asn Phe Lys Ile Arg His 165 170 175 aac atc gag gac ggc agc gtg cag ctc gcc gac cac tac cag cag aac 576 Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn 180 185 190 acc ccc atc ggc gac ggc ctc gtg ctg ctg ccc gac aac cac tac ctg 624 Thr Pro Ile Gly Asp Gly Leu Val Leu Leu Pro Asp Asn His Tyr Leu 195 200 205 agc tac cag tcc gcc ctg agc aaa gac ccc aac gag aag cgc gat cac 672 Ser Tyr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His 210 215 220 atg gtc ctg ctg gag ttc gtg acc gcc gcc ggg atc act atc ggc atg 720 Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Ile Gly Met 225 230 235 240 gac gag ctg tac aag taa 738 Asp Glu Leu Tyr Lys 245 <210> 4 <211> 245 <212> PRT <213> Aequorea victoria <400> 4 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 1 5 10 15 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 20 25 30 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 35 40 45 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50 55 60 Phe Gly Tyr Gly Leu Met Cys Phe Ala Arg Tyr Pro Asp His Met Lys 65 70 75 80 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 85 90 95 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100 105 110 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135 140 Asn Tyr Gly Gly Ser Gly Ala Ser Asn Ser His Asn Val Tyr Ile Met 145 150 155 160 Ala Asp Lys Gln Lys Asn Gly Ile Lys Val Asn Phe Lys Ile Arg His 165 170 175 Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn 180 185 190 Thr Pro Ile Gly Asp Gly Leu Val Leu Leu Pro Asp Asn His Tyr Leu 195 200 205 Ser Tyr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His 210 215 220 Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Ile Gly Met 225 230 235 240 Asp Glu Leu Tyr Lys 245<110> NEUROGENEX Co.Ltd. <120> Enhanced inserted YFP <160> 4 <170> Kopatent In 1.71 <210> 1 <211> 738 <212> DNA <213> Aequorea victoria <220> <221> CDS <222> (1) .. (735) <223> y-citrine DNA sequence <400> 1 atg gtg agc aag ggc gag gag ctg ttc acc ggg gtg gtg ccc atc ctg 48 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 1 5 10 15 gtc gag ctg gac ggc gac gta aac ggc cac aag ttc agc gtg tcc ggc 96 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 20 25 30 gag ggc gag ggc gat gcc acc tac ggc aag ct t acc atc 144 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 35 40 45 tgc acc acc ggc aag ctg ccc gtg ccc tgg ccc acc ctc gtg act acc 192 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50 55 60 ttc ggc tac ggc ctg atg tgc ttc gcc cgc tac ccc gac cac atg aag 240 Phe Gly Tyr Gly Leu Met Cys Phe Ala Arg Tyr Pro Asp His Met Lys 65 70 75 80 cag cac gac ttc ttc aag tcc gcc atg ccc gaa ggc tac gtc cag gag 288 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 85 90 95 cgc acc atc ttc ttc aag gac gac ggc aac tac aag acc cgc gcc gag 336 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100 105 110 gtg aag ttc gag ggc gac acc ctg gtg aac cgc atc gag ctg aag ggc 384 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125 atc gac ttc aag gag gac ggc aac atc ctg ggg cac aag ctg gag tac 432 Ile Asp Phe Lys Glu As p Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135 140 aac tac ggt gga tcc ggt gct agc aac agc cac aac gtc tat atc atg 480 Asn Tyr Gly Gly Ser Gly Ala Ser Asn Ser His Asn Val Tyr Ile Met 145 150 155 160 gcc gac aag cag aag aac ggc atc aag gtg aac ttc aag atc cgc cac 528 Ala Asp Lys Gln Lys Asn Gly Ile Lys Val Asn Phe Lys Ile Arg His 165 170 175 aac atc gag gac ggc agc gtg cag ctc gtac gcc cag cag aac 576 Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn 180 185 190 acc ccc atc ggc gac ggc ccc gtg ctg ctg ccc gac aac cac tac ctg 624 Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu 195 200 205 agc tac cag tcc gcc ctg agc aaa gac ccc aac gag aag cgc gat cac 672 Ser Tyr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His 210 215 220 atg gtc ctg ctg gag ttc gtg acc gcc gcc ggg atc act ctc ggc atg 720 Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met 225 230 235 240 gac gag ctg tac aag taa 738 Asp Glu Leu Tyr Lys 245 <210> 2 <211> 245 <212> PRT <213> Aequorea victoria <400> 2 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 1 5 10 15 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 20 25 30 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 35 40 45 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50 55 60 Phe Gly Tyr Gly Leu Met Cys Phe Ala Arg Tyr Pro Asp His Met Lys 65 70 75 80 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 85 90 95 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100 105 110 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135 140 Asn Tyr Gly Gly Ser Gly Ala Ser Asn Ser His Asn Val Tyr Ile Met 145 150 155 160 Ala Asp Lys Gln Lys Asn Gly Ile Lys Val Asn Phe Lys Ile Arg His 165 170 175 Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn 180 185 190 Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu 195 200 205 Ser Tyr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His 210 215 220 Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met 225 230 235 240 Asp Glu Leu Tyr Lys 245 <210> 3 <211> 738 <212> DNA <213> Aequorea victoria <220> <221> CDS <222> (1) .. (735) <223> Peridot DNA sequence <400> 3 atg gtg agc aag ggc gag gag ctg ttc acc ggg gtg gtg ccc atc ctg 48 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 1 5 10 15 gtc gag ctg gac ggc gac gta aac ggc cac aag ttc agc gtg tcc ggc 96 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 20 25 30 gag ggc gag ggc gat gcc acc tac ggc aag ctg acc ctg aag ttc atc 144 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 35 40 45 tgc acc acc ggc aag ctg ccc gtg ccc tgg ccc acc ctc gtg act acc 192 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50 55 60 ttc ggc tac ggc ctg atg tgc ttc gcc cgc tac ccc gac cac atg aag 240 Phe Gly Tyr Gly Leu Met Cys Phe Ala Arg Tyr Pro Asp His Met Lys 65 70 75 80 cag cac gac ttc ttc aag tcc gcc atg ccc gaa ggc tac gtc cag gag 288 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 85 90 95 cgc acc atc ttc ttc aag gac gac ggc aac tac aag acc cgc gcc gag 336 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100 105 110 gtg aag ttc gag ggc gac acc ctg gtg aac cgc atc gag ctg aag ggc 384 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Gly 115 120 125 atc gac ttc aag gag gac ggc aac atc ctg ggg cac aag ctg gag tac 432 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135 140 aac tac ggt gga tcc ggt gct ag ag ag a cac aac gtc tat atc atg 480 Asn Tyr Gly Gly Ser Gly Ala Ser Asn Ser His Asn Val Tyr Ile Met 145 150 155 160 gcc gac aag cag aag aac ggc atc aag gtg aac ttc aag atc cgc cac 528 Ala Asp Lys Gln Lys Asn Gly Ile Lys Val Asn Phe Lys Ile Arg His 165 170 175 aac atc gag gac ggc agc gt g cag ctc gcc gac cac tac cag cag aac 576 Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn 180 185 190 acc ccc atc ggc gac ggc ctc gtg ctg ctg ccc gac aac cac tac ctg 624 Thr Pro Ile Gly Asp Gly Leu Val Leu Leu Pro Asp Asn His Tyr Leu 195 200 205 agc tac cag tcc gcc ctg agc aaa gac ccc aac gag aag cgc gat cac 672 Ser Tyr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His 210 215 220 atg gtc ctg ctg gag ttc gtg acc gcc gcc ggg atc act atc ggc atg 720 Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Ile Gly Met 225 230 235 240 gac gag ctg tac aag taa 738 Asp Glu Leu Tyr Lys 245 <210> 4 <211> 245 <212> PRT <213> Aequorea victoria <400> 4 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 1 5 10 15 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 20 25 30 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 35 40 45 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50 55 60 Phe Gly Tyr Gly Leu Met Cys Phe Ala Arg Tyr Pro Asp His Met Lys 65 70 75 80 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 85 90 95 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100 105 110 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135 140 Asn Tyr Gly Gly Ser Gly Ala Ser Asn Ser His Asn Val Tyr Ile Met 145 150 155 160 Ala Asp Lys Gln Lys Asn Gly Ile Lys Val Asn Phe Lys Ile Arg His 165 170 175 Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn 180 185 190 Thr Pro Ile Gly Asp Gly Leu Val Leu Leu Pro Asp Asn His Tyr Leu 195 200 205 Ser Tyr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His 210 215 220 Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Ile Gly Met 225 230 235 240 Asp Glu Leu Tyr Lys 245

Claims

Insert fluorescent protein having the amino acid sequence of SEQ ID NO: 2.

Gene base sequence of SEQ ID NO: 1 encoding the fluorescent protein of claim 1.

delete

The fluorescent protein of claim 1, wherein amino acid No. 192 is substituted with lysine in proline (P192L).

Gene base sequence of SEQ ID NO: 3 encoding the fluorescent protein of claim 4.

delete

The insertion type fluorescent protein according to claim 1 or 4, wherein a cleavage site of NS3 protease (HCV NS3) of human hepatitis C virus is inserted.

The gene base sequence of claim 2 or 5, wherein a restriction enzyme recognition site of BamHI and NheI is inserted for gene cloning.

A method for analyzing the activity of hepatitis C virus protease using the inserted fluorescent protein of claim 7 as a substrate of a protease.