KR970043041A - Isolation, Purification Methods, and the cDNA Gene Encoding Suspension Thrombolytic Enzyme - Google Patents

Isolation, Purification Methods, and the cDNA Gene Encoding Suspension Thrombolytic Enzyme Download PDF

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KR970043041A
KR970043041A KR1019950067279A KR19950067279A KR970043041A KR 970043041 A KR970043041 A KR 970043041A KR 1019950067279 A KR1019950067279 A KR 1019950067279A KR 19950067279 A KR19950067279 A KR 19950067279A KR 970043041 A KR970043041 A KR 970043041A
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protein
fractions
lbti
affinity
column
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KR100294149B1 (en
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최의성
이상기
권수진
최윤주
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장용택
신풍제약 주식회사
김은영
한국과학기술연구원
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6402—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals
    • C12N9/6405—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals not being snakes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/18—Ion-exchange chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/20—Partition-, reverse-phase or hydrophobic interaction chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes

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  • General Engineering & Computer Science (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

본 발명은 구인(Earthworm)의 조추출물로부터 혈전용해효소를 순수하게 분리, 정제하는 방법 및 이들 구인의 혈전용해효소를 코드화하는 cDNA 유전자에 관한 것이다.The present invention relates to a method for purely separating and purifying thrombolytic enzymes from crude extracts of Earthworm, and to cDNA genes encoding thrombolytic enzymes of these antigens.

Description

구인 혈전용해효소의 분리, 정제방법 및 구인 혈전용해효소를 코드화하는 cDNA유전Isolation, Purification Methods, and cDNA Genes Encoding Sump Thrombolytic Enzymes

본 내용은 요부공개 건이므로 전문내용을 수록하지 않았음Since this is an open matter, no full text was included.

제 1도는 3' -RACE 및 5' -RACE에 의한 전체(full lengh) cDNA 유전자의 분리과정을 도식화하여 나타낸 것이다.FIG. 1 is a schematic diagram showing the isolation process of full lengh cDNA gene by 3'-RACE and 5'-RACE.

제 2도는 구인 조추출물을 DEAE 음이온 교환 칼럼에 적용하여 얻은 크로마토그램이다.2 is a chromatogram obtained by applying the crude crude extract to a DEAE anion exchange column.

제 3도의 A는 음이온 교환 칼럼 크로마토그래피를 통해 얻어진 여섯 분획 중 D(Ⅰ)을 페닐-세파로오스 소수성 결합 칼럼에 적용하여 얻은 크로마토그램이고, B는 분획 D(Ⅱ)를 동일 칼럼에 적용하여 얻은 크로마토그램이다.A of FIG. 3 is a chromatogram obtained by applying D (I) of the six fractions obtained through anion exchange column chromatography to a phenyl-sepharose hydrophobic binding column, and B is applying fraction D (II) to the same column. Obtained chromatogram.

Claims (17)

혈전용해효소 P1을 코드화하는 하기 서열의 cDNA 유전자 LK7T1(등록번호 U25645).CDNA gene LK7T1 (registration no. U25645) of the following sequence encoding a thrombolytic enzyme P1. GGACTGATCCTGGAATTTGTTACTTCAGGCTACACAGTAACACAAGATGCGTTCATTTGTGGCTTTTCTCGCTGCTCTCAGCCTTTGCCAGGCGAGGCCCCAGAAATTCCTCGATGGTGCCCGTCCATCCTTCAGGATGGGTGGTGAGCAGTACATCATCGGTGGATCGAACGCTAGTCCAGGACAGTTTCCATGGCAACTGTCTCAGACACGAGGAGGATCACACAGCTGCGGCGCTTCACTGCTCAACGCACTCAACGGTCTCAGTGCATTTCACTGCGTCGATGGAGCAGCTCCCGGCACCATCACAGTAATCGCTGGTCTGCATGATCGATCGGGCACACCTGGATCTCAGGAAGTAGACATAACAGGATACACGATGCACGAGAATTATAACCAGGGTACTAATACCTATGCCAATGACATAGCCATTCTGCACTTTGCATCGGCAATAAACATTGGAGGTAACGGTCAGGCGGCTCTCCTTCCAGCGAACAACGACAACGACTACAGCGGCCTTACTTGCGTCATTTCAGGATGGGGCCGGAAACCTTCGTCCAATGTCCTTCCTGATACTCTTCAGAAGGCATCCATTCAAGTCATCGGTACTACTCAGTGCCAGAGTCTTATGGGAAGCATCGGCCACATTTGGGATAACCACATCTGCCTCTACAACAATACTAATACGTTGGATCCTGCAACGGTGATAGCGGTGGCCCGTTGACTGCCCAGACGGTGGTACTCGCGTTGCTGGTGTTACTTCATGGGGTGTATCGAGTGGAGCTGGCAACTGTCTTCAGACATACCCAACCGTTTACACCCGCACAAGCGCCTACCTGTCTTGGATCGCAAACAACTCTTAGTAAGAAGGGATCACGTTATCACGACAACGATTGGAAACCAGAAACATTTACCCTTTACAAAAGTCAATAATAGATGGAAGAGACTAGGCATAAAGAGTGTCGTTTTCACCTGATGAGAACTAAATTGATGGCTATTGACTTGTTGTAATTTGAAAGAGTAAATAACGTCTGCAACAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGGACTGATCCTGGAATTTGTTACTTCAGGCTACACAGTAACACAAGATGCGTTCATTTGTGGCTTTTCTCGCTGCTCTCAGCCTTTGCCAGGCGAGGCCCCAGAAATTCCTCGATGGTGCCCGTCCATCCTTCAGGATGGGTGGTGAGCAGTACATCATCGGTGGATCGAACGCTAGTCCAGGACAGTTTCCATGGCAACTGTCTCAGACACGAGGAGGATCACACAGCTGCGGCGCTTCACTGCTCAACGCACTCAACGGTCTCAGTGCATTTCACTGCGTCGATGGAGCAGCTCCCGGCACCATCACAGTAATCGCTGGTCTGCATGATCGATCGGGCACACCTGGATCTCAGGAAGTAGACATAACAGGATACACGATGCACGAGAATTATAACCAGGGTACTAATACCTATGCCAATGACATAGCCATTCTGCACTTTGCATCGGCAATAAACATTGGAGGTAACGGTCAGGCGGCTCTCCTTCCAGCGAACAACGACAACGACTACAGCGGCCTTACTTGCGTCATTTCAGGATGGGGCCGGAAACCTTCGTCCAATGTCCTTCCTGATACTCTTCAGAAGGCATCCATTCAAGTCATCGGTACTACTCAGTGCCAGAGTCTTATGGGAAGCATCGGCCACATTTGGGATAACCACATCTGCCTCTACAACAATACTAATACGTTGGATCCTGCAACGGTGATAGCGGTGGCCCGTTGACTGCCCAGACGGTGGTACTCGCGTTGCTGGTGTTACTTCATGGGGTGTATCGAGTGGAGCTGGCAACTGTCTTCAGACATACCCAACCGTTTACACCCGCACAAGCGCCTACCTGTCTTGGATCGCAAACAACTCTTAGTAAGAAGGGATCACGTTATCACGACAACGATTGGAAACCAGAAACATTTACCCTTTACAAAAGTCAATAATAGATGGAAGAGACTAGGCATAAAGAGTGTCGTTTTCACCTGATGAGAACTAAATTGATGGCTATTG ACTTGTTGTAATTTGAAAGAGTAAATAACGTCTGCAACAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 혈전용해호소 P2를 코드화하는 하기 서열의 cDNA 유전자 LKP2(등록번호 U36338).CDNA gene LKP2 (registration no. U36338) of the following sequence, which encodes thrombolytic P2. TGAATTCGTTTTGTTCTACGAAAGAATTCAATCGTAAACCATGAGGATCCTGCTGCTTTTGTGTTTGGCTCTGCCAGCTTTCGGGAGCGGAGTGATCAGCTGTTGTCGGTGGAAGTGATACCACGATCGGCCAATACCCGCATCAGCTGTCGCTGCGGTGACTGGTTCTCACTCATGCGGAGCATCTCTGATTGGAACCACTCGAGCGGTTACCGCGGCTCACTGCACAGGATCTGCCATCGGCGTCTACACTATTCTTGGCGGTACCACTGATCGAACGGTTACAAACTGTGCCACCTGTGTCCTCAGAGATCTGAACTTCTTGAACAGGCATCCTCAGTACGATGAGAATGGAAACGGATATCCAAATGACGTAGCCGTTATTGGTTTCGCCGCTGTCGCCACCAACACCAACCTCCAGACGATAAGCCTGGCCACTCCATCAGACGGAAGCTTTGCTGGTGACACTGGCGTCATCACCGGTTGGGGCAAGACTGCGTCTATTGGAGGTATTCCGGACATCTTGCAAATGGCTACGATGAATGTTATCACCAACGCGGATTGTGCTGGTACTTGGGGCGCACTTAGCATCAACGACGGACACATCTGCGTCTCTGCTGTCGGGAGAAGCGCCTGCAGTGGTGACAGCGGTGGTCCTCTGGAGTGCGGTAATACCCTGGCTGGAGCAACATCTTGGGGCCAGGCCTCGTGTGACCCATCCTACCCGTCCGTCTACACAAGAATCAGCTACTTCTACTCCTGGATCATCGCGCAATAAGCAACGCGACGACTTAATTCTAAATTGCCTTAACGCTATTTCTGCTTGCAATTAAGCAACAAGAAATGATACACGAACATAATATATATTAGACTTGCATGACAGTCGTTAAATAAACTGTCAACCTCGAAAAAAAAAAAAAAAAAAATGAATTCGTTTTGTTCTACGAAAGAATTCAATCGTAAACCATGAGGATCCTGCTGCTTTTGTGTTTGGCTCTGCCAGCTTTCGGGAGCGGAGTGATCAGCTGTTGTCGGTGGAAGTGATACCACGATCGGCCAATACCCGCATCAGCTGTCGCTGCGGTGACTGGTTCTCACTCATGCGGAGCATCTCTGATTGGAACCACTCGAGCGGTTACCGCGGCTCACTGCACAGGATCTGCCATCGGCGTCTACACTATTCTTGGCGGTACCACTGATCGAACGGTTACAAACTGTGCCACCTGTGTCCTCAGAGATCTGAACTTCTTGAACAGGCATCCTCAGTACGATGAGAATGGAAACGGATATCCAAATGACGTAGCCGTTATTGGTTTCGCCGCTGTCGCCACCAACACCAACCTCCAGACGATAAGCCTGGCCACTCCATCAGACGGAAGCTTTGCTGGTGACACTGGCGTCATCACCGGTTGGGGCAAGACTGCGTCTATTGGAGGTATTCCGGACATCTTGCAAATGGCTACGATGAATGTTATCACCAACGCGGATTGTGCTGGTACTTGGGGCGCACTTAGCATCAACGACGGACACATCTGCGTCTCTGCTGTCGGGAGAAGCGCCTGCAGTGGTGACAGCGGTGGTCCTCTGGAGTGCGGTAATACCCTGGCTGGAGCAACATCTTGGGGCCAGGCCTCGTGTGACCCATCCTACCCGTCCGTCTACACAAGAATCAGCTACTTCTACTCCTGGATCATCGCGCAATAAGCAACGCGACGACTTAATTCTAAATTGCCTTAACGCTATTTCTGCTTGCAATTAAGCAACAAGAAATGATACACGAACATAATATATATTAGACTTGCATGACAGTCGTTAAATAAACTGTCAACCTCGAAAAAAAAAAAAAAAAAAA 혈전용해효소 P3를 코드화하는 하기 서열의 cDNA 유전자 LK3(1) (등록번호 U25648).CDNA gene LK3 (1) (registration no. U25648) of the following sequence encoding a thrombolytic enzyme P3. GGAGTCGAGTCTTTAAATCAGCAAGATGTTACTTCTCGCTCTTGCATCGCTCGTAGCGGTGGGCTTTGCCCAACCACCAATCTGGTACCCCGGTGGTCAATGCGGTCTCAGCCAGTACTCAGATGCTGGTGACATGGAACTTCCTCCCGGAACAAAAATTGTCGGAGGAATTCAAGCTAGACCATACGAGTTCCCGTGGCAGGTGTCCGTCCGAAGGAAGTCTTCCGATTCCCATTTCTGCGGAGGTAGCATCATCAACGATCGTTGGGTTGTCTGCGCTGCTCACTGCATGCAGGGAGAGGCCCCCGCTCTGGTTTCATTGGTCGTGGGTGAGCACGACAGGAGTGCAGCGAGTGCAGTACGTCAGACTCATGACGTTGATAGCATCTTCGTTCACGAGGACTACACACAAATACCCTAGAGAACGACGTTTCTGTCATCAAGACATCTGTTGCCATCACTTTCGACATCAACGTTGGTCCAATCTGTGCCCCAGATCCAGCTCAACAGTACGTCTACCGTAAGAGCCAGTGCTCCGGATGGGGAACTATCAATTCAGGTGGAATCTGCTGTCCCAACATTCTGCGATACGTGACGCTGAATGTCACAACCAACCAATTCTGCGAAGATGTATACCCACTAAATTCAATCTTCGACGATATGATTTGCGCGTCGGACAACACCGGGGGTAACGACAGAGACTCCTGCCAGGGTGACTCCGGCGGCCCTCTGAGCGTCAAGGATGGCAGTGGAATCTTCAGCCTGATTGGTATTGTGTCTTGGGGAATTGGTTGCGCTTCTGGCTATCCAGGAGTCTACTCCCGCGTCGGATTCCATACTGCATGGATCACCGACATCATCACCAACAACTAAACCGGAGTTATTCAGTCGACTATAACTGGAACGACTTTACCATCACGAACGACACATTAATTGGAGTCGAGTCTTTAAATCAGCAAGATGTTACTTCTCGCTCTTGCATCGCTCGTAGCGGTGGGCTTTGCCCAACCACCAATCTGGTACCCCGGTGGTCAATGCGGTCTCAGCCAGTACTCAGATGCTGGTGACATGGAACTTCCTCCCGGAACAAAAATTGTCGGAGGAATTCAAGCTAGACCATACGAGTTCCCGTGGCAGGTGTCCGTCCGAAGGAAGTCTTCCGATTCCCATTTCTGCGGAGGTAGCATCATCAACGATCGTTGGGTTGTCTGCGCTGCTCACTGCATGCAGGGAGAGGCCCCCGCTCTGGTTTCATTGGTCGTGGGTGAGCACGACAGGAGTGCAGCGAGTGCAGTACGTCAGACTCATGACGTTGATAGCATCTTCGTTCACGAGGACTACACACAAATACCCTAGAGAACGACGTTTCTGTCATCAAGACATCTGTTGCCATCACTTTCGACATCAACGTTGGTCCAATCTGTGCCCCAGATCCAGCTCAACAGTACGTCTACCGTAAGAGCCAGTGCTCCGGATGGGGAACTATCAATTCAGGTGGAATCTGCTGTCCCAACATTCTGCGATACGTGACGCTGAATGTCACAACCAACCAATTCTGCGAAGATGTATACCCACTAAATTCAATCTTCGACGATATGATTTGCGCGTCGGACAACACCGGGGGTAACGACAGAGACTCCTGCCAGGGTGACTCCGGCGGCCCTCTGAGCGTCAAGGATGGCAGTGGAATCTTCAGCCTGATTGGTATTGTGTCTTGGGGAATTGGTTGCGCTTCTGGCTATCCAGGAGTCTACTCCCGCGTCGGATTCCATACTGCATGGATCACCGACATCATCACCAACAACTAAACCGGAGTTATTCAGTCGACTATAACTGGAACGACTTTACCATCACGAACGACACATTAATT 혈전용해효소 P4를 코드화하는 하기 서열의 cDNA 유전자 LKDa2 (등록번호 U25647).The cDNA gene LKDa2 (registration no. U25647) of the following sequence encoding a thrombolytic enzyme P4. GATCCCAGATTACTTCATCGAAGAAAATGAGGTCGATCGTCGCAGTCCTTGCAGCGCTCACCCTGTGTCAGGCCAAGCCACATGTTCGCTTGGGTGGCCCTGGCGTGTCATTAGTCAGGAATGCTAACCCATACGTCATTGGTGGAACCAACGCTAGCCCAGGAGAGTTCCCGTGGCAACTGTCTCAGCAACGACAAAGCGGTTCTTGGTCGCACAGCTGCGGAGCATCGCTTCTGAGCTCAACTTCCGCTCTCAGCGCATCGCACTGCGTCGATGGAGTGCTACCCAACAACATTCGAGTCATCGCTGGTCTTTGGCAACAGTCGGAAACAAGCGGCACTCAGACCGCTAACGTCGATAGCTACACCATGCATGAGAACTATGGTGCTGGTACTGCTTCGTACTCCAATGACATTGCCATTCTGCACCTCGCAACGTCCATCAGCCTCGGAAGAAACTCCAGGCAGCTGTCCTTCCCGCCAACAACAACAACGACTACGCCGGAACCACATGCGTCATCTCCGGATGGGGTCGCACAGATGGAACGAACAATCCGCCGGACATCCTTCAGAAGTCGTCAATTCCGGTCATTACGACCGCCCAGTGCACCGCCGCCATGGTTGGAGTCGGTGGAGCCAACATCTGGGATAATCACATCTGCGTCCAGGATCCTGCTGGCAACACCGGAGCCTGCAATGGCGATAGCGGTGGCCCACTGAACTGCCCAGACGGCGGAACTCGAGTGGTTGGTGTTACTTCGTGGGTTGTATCCAGTGGCCTTGGTGCATGTCTTCCGGACTACCCTTCCGTCTACACCCGCGTCAGGGCTTACTTGGGTTGGATCGGAGACAACTCTCGTTGGAGAGTCACACTGATAAAGAAAGCTCCAGAAAAACAAGCAGTCAACATCTCGAAAACAGAAGTTATACGGACTTACAAGTGGCATTCCAATAATTATTGTCACCATAATAACGATTTAATGCAGGCGTAAAAGCTGGTTCTCATTAAATCCGTTCATATCAAAAAAAAAACAAAAAAAAAAAAAGATCCCAGATTACTTCATCGAAGAAAATGAGGTCGATCGTCGCAGTCCTTGCAGCGCTCACCCTGTGTCAGGCCAAGCCACATGTTCGCTTGGGTGGCCCTGGCGTGTCATTAGTCAGGAATGCTAACCCATACGTCATTGGTGGAACCAACGCTAGCCCAGGAGAGTTCCCGTGGCAACTGTCTCAGCAACGACAAAGCGGTTCTTGGTCGCACAGCTGCGGAGCATCGCTTCTGAGCTCAACTTCCGCTCTCAGCGCATCGCACTGCGTCGATGGAGTGCTACCCAACAACATTCGAGTCATCGCTGGTCTTTGGCAACAGTCGGAAACAAGCGGCACTCAGACCGCTAACGTCGATAGCTACACCATGCATGAGAACTATGGTGCTGGTACTGCTTCGTACTCCAATGACATTGCCATTCTGCACCTCGCAACGTCCATCAGCCTCGGAAGAAACTCCAGGCAGCTGTCCTTCCCGCCAACAACAACAACGACTACGCCGGAACCACATGCGTCATCTCCGGATGGGGTCGCACAGATGGAACGAACAATCCGCCGGACATCCTTCAGAAGTCGTCAATTCCGGTCATTACGACCGCCCAGTGCACCGCCGCCATGGTTGGAGTCGGTGGAGCCAACATCTGGGATAATCACATCTGCGTCCAGGATCCTGCTGGCAACACCGGAGCCTGCAATGGCGATAGCGGTGGCCCACTGAACTGCCCAGACGGCGGAACTCGAGTGGTTGGTGTTACTTCGTGGGTTGTATCCAGTGGCCTTGGTGCATGTCTTCCGGACTACCCTTCCGTCTACACCCGCGTCAGGGCTTACTTGGGTTGGATCGGAGACAACTCTCGTTGGAGAGTCACACTGATAAAGAAAGCTCCAGAAAAACAAGCAGTCAACATCTCGAAAACAGAAGTTATACGGACTTACAAGTGGCATTCCAATAATTATTGTCACCATAATAACGATTTAATGCAGGCGTAAAAGCTGGTT CTCATTAAATCCGTTCATATCAAAAAAAAAACAAAAAAAAAAAAA 혈전용해효소 P6를 코드화하는 하기 서열의 cDNA 유전자 LK7T2 (등록번호 U25646).CDNA gene LK7T2 (registration no. U25646) of the following sequence encoding a thrombolytic enzyme P6. TGACTGATCTTGGAATTTGTTACACCAGGCTACCAGTAATACAAGATGCGTTCATTTGTGGCTTTTCTTGCCGCTATAAGCCTGTGTAAGGCGAGGTCACAGTCGTTCATCGATGGTGCCCGTCCGTCTTTCATGATGGGTGGTGAGCAGTACGTCATCGGTGGAACGGACGCTGCTCCAGGACACTTTCCATGGCAACTGTCTCAGACACGTGGAGGATCGCACAGTTGCGGCGCTTCACTGCTCAGCTCGAATTCCGGTCTTAGTGCATCTCACTGCGTCGATGGAGCACTTCCAGGCTCCATCACAGTAATCGCTGGTCTGCATGATCGATCGGGCACACCTGGATCTCAGGAAGTAGACATCACAGGATACACGATGCACGAGGAATACCTGACGGGTATTTATACCTATTCTAATGACATATCCATTCTGAACTTTGCAACGCCAATAACCATTGGAGGCAATATTCAGCCGGCCACCCTTCCAGCGGACAACAGCAACAACTACCTCGGCCTTACTTGCGTCATTTCTGGACGGGGCCAAACAAGTTCGTCCAATATCCTTCCTGACACCCTTCAGAAGGCATCCATTCAAGTCATCGGTACTGATGAGTGCCACACTCTTGTCGATAACGTCCTCGGTTACAAGATTTGGCATAACCATATCTGTATCTACAACCAGACTAATAGCGTTGGATCATGCAACGGTGATAGCGGTGGCCCATTGAACTGCCCAGACGGTACTACCGTCGTTGCTGGTATTACGTCATGGGGAATATCGAGTGGAGGCGACTGTCTCCAGGATTACCCATCCGTTTACACCCGCACAAGCGCCTACCTGGATTGGATCGCAGCCAACTCTTCGTAAGAAGGGATCATGTTCTCACGACAACGATTGGAGGCAAGGAACAATTGCCGTTTACACAAGACCACAATAGATGGAAGAGATTAAAGAGACTGTCGCTTTCACCTGATGAGAACTGAATTTATGGCTATTGACTTGTTGTAATTTGAAAGAATAAATGAACGTCAACAAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATGACTGATCTTGGAATTTGTTACACCAGGCTACCAGTAATACAAGATGCGTTCATTTGTGGCTTTTCTTGCCGCTATAAGCCTGTGTAAGGCGAGGTCACAGTCGTTCATCGATGGTGCCCGTCCGTCTTTCATGATGGGTGGTGAGCAGTACGTCATCGGTGGAACGGACGCTGCTCCAGGACACTTTCCATGGCAACTGTCTCAGACACGTGGAGGATCGCACAGTTGCGGCGCTTCACTGCTCAGCTCGAATTCCGGTCTTAGTGCATCTCACTGCGTCGATGGAGCACTTCCAGGCTCCATCACAGTAATCGCTGGTCTGCATGATCGATCGGGCACACCTGGATCTCAGGAAGTAGACATCACAGGATACACGATGCACGAGGAATACCTGACGGGTATTTATACCTATTCTAATGACATATCCATTCTGAACTTTGCAACGCCAATAACCATTGGAGGCAATATTCAGCCGGCCACCCTTCCAGCGGACAACAGCAACAACTACCTCGGCCTTACTTGCGTCATTTCTGGACGGGGCCAAACAAGTTCGTCCAATATCCTTCCTGACACCCTTCAGAAGGCATCCATTCAAGTCATCGGTACTGATGAGTGCCACACTCTTGTCGATAACGTCCTCGGTTACAAGATTTGGCATAACCATATCTGTATCTACAACCAGACTAATAGCGTTGGATCATGCAACGGTGATAGCGGTGGCCCATTGAACTGCCCAGACGGTACTACCGTCGTTGCTGGTATTACGTCATGGGGAATATCGAGTGGAGGCGACTGTCTCCAGGATTACCCATCCGTTTACACCCGCACAAGCGCCTACCTGGATTGGATCGCAGCCAACTCTTCGTAAGAAGGGATCATGTTCTCACGACAACGATTGGAGGCAAGGAACAATTGCCGTTTACACAAGACCACAATAGATGGAAGAGATTAAAGAGACTGTCGCTTTCACCTGATGAGAACTGAATTTATGGCTATTG ACTTGTTGTAATTTGAAAGAATAAATGAACGTCAACAAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 혈전용해효소 P7을 코드화하는 하기 서열의 cDNA 유전자 LK1T4 (등록번호 U25643).CDNA gene LK1T4 (registration no. U25643) of the following sequence encoding a thrombolytic enzyme P7. TCTCGAGTCTATCATTAAGATGTTACTTCTCGCCCTTGCATCGTTGGTAGCGGTGGGTTTTGCGCAACCACCAGTCTGGTACCCCGGTGGTCAATGCAGTGTCAGCCAGTACTCAGATGCTGGTGACATGGAACTTCCTCCCGGAACAAAAATTGTCGGAGGAATTGAAGCCAGACCATACGAGTTCCCATGGCAGGTGTCCGTCCGAAGGAAGTCTTCCGATTCCCATTTCTGCGGAGGTAGCATCATCAACGATCGTTGGGTTGTCTGCGCTGCTCACTGCATGCAGGGAGAGAGCCCTGCCCTGGTTTCATTGGTCGTCGGTGAGCACGATAGCAGCGCTGCGAGTACAGTACGTCAGACTCATGACGTTGACAGCATCTTCGTCCACGAGGACTACAACGGAAATACCTTTGAGAACGACGTTTCTGTCATCAAGACAGTTAACGCCATCGCCATCGACATCAACGATGGGCCAATCTGCGCTCCAGATCCAGCCAACGATTACGTCTACCGTAAGAGCCAGTGCTCCGGATGGGGAACTATCAACTCAGGTGGAGTCTGCTGCCCCAACGTTCTGCGATATGTGACACTGAACGTCACAACCAACGCCTTCTGCGATGATATCTACAGCCCATTATATACAATTACCAGCGACATGATCTGCGCCACGGACAACACCGGACAGAACGAGAGAGACTCTTGCCAGGGTGACTCTGGCGGCCCTCTGAGCGTCAAGGATGGCAACGGAATCTTCAGCCTCATTGGTATTGTGTCTTGGGGAATCGGTTGCGCATCTGGCTATCCAGGAGTCTACGCCCGCGTCGGATCCCAAACTGGATGGATCACAGACATTATTACCAACAACTAGACCGGACTTAGCCAGTCGATTATAGCTGGAACGACTCTATTATAACGAATGACACATTTATTAACATTATTGTGGTAACATGGAGTAAAATCTCTCTTCTCNCNANAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATCTCGAGTCTATCATTAAGATGTTACTTCTCGCCCTTGCATCGTTGGTAGCGGTGGGTTTTGCGCAACCACCAGTCTGGTACCCCGGTGGTCAATGCAGTGTCAGCCAGTACTCAGATGCTGGTGACATGGAACTTCCTCCCGGAACAAAAATTGTCGGAGGAATTGAAGCCAGACCATACGAGTTCCCATGGCAGGTGTCCGTCCGAAGGAAGTCTTCCGATTCCCATTTCTGCGGAGGTAGCATCATCAACGATCGTTGGGTTGTCTGCGCTGCTCACTGCATGCAGGGAGAGAGCCCTGCCCTGGTTTCATTGGTCGTCGGTGAGCACGATAGCAGCGCTGCGAGTACAGTACGTCAGACTCATGACGTTGACAGCATCTTCGTCCACGAGGACTACAACGGAAATACCTTTGAGAACGACGTTTCTGTCATCAAGACAGTTAACGCCATCGCCATCGACATCAACGATGGGCCAATCTGCGCTCCAGATCCAGCCAACGATTACGTCTACCGTAAGAGCCAGTGCTCCGGATGGGGAACTATCAACTCAGGTGGAGTCTGCTGCCCCAACGTTCTGCGATATGTGACACTGAACGTCACAACCAACGCCTTCTGCGATGATATCTACAGCCCATTATATACAATTACCAGCGACATGATCTGCGCCACGGACAACACCGGACAGAACGAGAGAGACTCTTGCCAGGGTGACTCTGGCGGCCCTCTGAGCGTCAAGGATGGCAACGGAATCTTCAGCCTCATTGGTATTGTGTCTTGGGGAATCGGTTGCGCATCTGGCTATCCAGGAGTCTACGCCCGCGTCGGATCCCAAACTGGATGGATCACAGACATTATTACCAACAACTAGACCGGACTTAGCCAGTCGATTATAGCTGGAACGACTCTATTATAACGAATGACACATTTATTAACATTATTGTGGTAACATGGAGTAAAATCTCTCTTCTCNCNANAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAA 음이온 교환 칼럼 크로마토그래피, 소수성 결합 칼럼 크로마토그래피, 벤자미딘을 리간드로 사용하는 친화성 크로마토그래피 및 리마빈 트립신 억제제(Lima Bean Trypsin Inhibitor ; LBTI)를 리간드로 사용하는 친화성 크로마토그래피의 조합에 의하여 구인 (Earthworm)의 조추출물로부터 혈전용해활성을 갖는 효소를 분리, 정제하는 방법.By a combination of anion exchange column chromatography, hydrophobic binding column chromatography, affinity chromatography using benzamidine as a ligand and affinity chromatography using Lima Bean Trypsin Inhibitor (LBTI) as a ligand A method for separating and purifying enzymes having thrombolytic activity from crude extracts of Earthworm. 제 7항에 있어서, 음이온 교환 칼럼이 DEAE Toyopearl-650(s)인 방법.8. The method of claim 7, wherein the anion exchange column is DEAE Toyopearl-650 (s). 제 7항에 있어서, 소수성 결합 칼럼이 페닐-세파로오스를 사용한 것인 방법.8. The method of claim 7, wherein the hydrophobic binding column uses phenyl-sepharose. 제 7항에 있어서, 구인이 붉은 지렁이 룸브리쿠스 루벨루스(Lumbricus rubellus)인 방법.8. The method of claim 7, wherein the job is a red earthworm Lumbricus rubellus. 제 7항 내지 10항 중의 어느 한 항에 있어서, (a) 구인의 조추출물을 DEAE Toyopearl-650(s)컬럼에 적용하여 혈전용해활성을 갖는 여섯가지 분획 D(Ⅰ), D(Ⅱ), D(Ⅲ), D(Ⅳ), D(Ⅴ) 및 D(Ⅵ)를 얻고, (b) (a)에서 수득한 분획 D(Ⅰ) 및 D(Ⅱ)를 각각 페닐-세파로오스 소수성 결합 칼럼에 적용하여 P1 및 P2 단백질 분획을 얻는 한편, 분획 D(Ⅲ), D(Ⅴ), D(Ⅵ)를 각각 멘자미딘-친화성 칼럼에 적용하고 흡착된 성분을 1M 아르기닌 용액으로 용출시켜 순수하게 정제된 P3 및 P7 단백질 분획을 얻으며, (c) (b)에서 수득한 P1 및 P2 단백질 분획을 LBTI-친화성 칼럼에 적용하고 흡착된 성분을 pH쇼크에 의해 용출시켜 순수하게 정제된 P1 및 P2 단백질 분획을 얻고, (d) (b)과정에서 D(Ⅲ) 및 D(Ⅴ) 분획을 벤자미딘-친화성 칼럼에 적용하였을 때 비흡착된 분획을 각각 LBTI-친화성 칼럼에 적용하여 P4 및 P6 단백질 분획을 얻음으로써 혈전용해활성을 갖는 효소 P1, P2, P3, P4, P6 및 P7을 분리, 정제하는 방법.(A) Six fractions D (I), D (II), having thrombolytic activity by applying crude extract of the strain to the DEAE Toyopearl-650 (s) column; D (III), D (IV), D (V) and D (VI), and (b) the fractions D (I) and D (II) obtained in (a), respectively, are phenyl-sepharose hydrophobic bonds. P1 and P2 protein fractions were applied to the column, while fractions D (III), D (V) and D (VI) were applied to the menzamidine-affinity column, respectively, and the adsorbed components were eluted with 1M arginine solution. Obtaining purely purified P3 and P7 protein fractions, (c) applying the P1 and P2 protein fractions obtained in (b) to an LBTI-affinity column and eluting the adsorbed components by pH shock to purely purified P1 And (2) applying the nonadsorbed fraction to the LBTI-affinity column, when (d) the D (III) and D (V) fractions were applied to the benzamidine-affinity column in (b). P4 P6 by obtaining the protein fraction separating enzyme P1, P2, P3, P4, P6 and P7 having a fibrinolytic activity, a method of purification. 제 7항에 있어서, 구인의 조추출물이 구인의 분쇄액을 30% 암모늄설페이트로 처리하여 침전물을 제거하고 여기서 얻어진 상등액을 다시 60% 암모늄설페이트로 처리하여 활성단백질이 포함되어 있는 침전물을 얻음으로써 제조된 것인 방법.The method of claim 7, wherein the crude extract of the gut is prepared by treating the ground liquor with 30% ammonium sulphate to remove the precipitate and treating the supernatant obtained again with 60% ammonium sulphate to obtain a precipitate containing the active protein. How it is. 벤자미딘을 리간드로 사용하는 친화성 크로마토그래피 및 리마빈 트립신 억제제(LBTI)를 리간드로 사용하는 친화성 크로마토그래피를 순차적으로 수행하여 구인 (Lumbricus rubellus)의 조추출물로부터 혈전용해활성을 갖는 효소를 분리, 정제하는 방법.An affinity chromatography using benzamidine as a ligand and an affinity chromatography using limabin trypsin inhibitor (LBTI) as a ligand were performed sequentially to obtain enzymes having thrombolytic activity from crude extracts of Lumbricus rubellus. Separation, Purification Method. 제 13항에 있어서, (a)구인의 조추출물을 벤자마딘-친화성 칼럼에 적용하여 흡착된 성분으로부터 순수하게 정제된 P3 및 P7 단백질 분획을 수득하고, (b) (a)과정에서 비흡착된 분획을 합해서 LBTI-친화성 칼럼에 적용하고 LBTI에 대한 친화정도에 따라 순수하게 정제된 P1,P2,P4 및 P6단백질 분획을 수득함으로써 혈전용해 활성을 갖는 효소 P1, P2, P3, P4, P6, P7을 분리, 정제하는 방법.The process of claim 13, wherein (a) crude extracts of ginseng are applied to a benzamadine-affinity column to obtain purified P3 and P7 protein fractions from the adsorbed components, and (b) The adsorbed fractions were combined and applied to the LBTI-affinity column and the purified P1, P2, P4 and P6 protein fractions were obtained according to their affinity for LBTI to obtain enzymes P1, P2, P3, P4, Method for separating and purifying P6 and P7. 제 14항에 있어서, (b)과정에서 얻어진 단백질 분획이 다른 단백질과 일부 혼합된 상태로 용출되는 경우 다시 합하여 LBTI-친화성 칼럼에 적용하는 과정을 반복함으로써 분리, 정제하는 방법.15. The method of claim 14, wherein when the protein fraction obtained in step (b) is eluted in a partially mixed state with another protein, the method is separated and purified by repeating the application of the protein fraction to the LBTI-affinity column. 제 14항 또는 15항에 있어서, 벤자미딘-친화성 칼럼 크로마토그래피에서 1M NaCl을 함유하는 50mM 아세트산나트륨 용액(pH 5.0)으로 세척하고 아르기닌 0에서 1M까지의 농도구배를 통해 흡착된 단백질을 용출시키는 방법.16. The protein of claim 14 or 15, washed with 50 mM sodium acetate solution (pH 5.0) containing 1 M NaCl in benzamidine-affinity column chromatography and eluting the adsorbed protein through a concentration gradient from arginine 0 to 1 M. How to let. 제 14항 또는 15항에 있어서, LBTI-친화성 칼럼 크로마토그래피에서 1M NaCl을 함유하는 50mM 아세트산나트륨 용액(pH 5.0)으로 세척하고 pH 2.8의 0.2M 글리신 완충용액으로 pH 쇼크를 주어 흡착된 단백질을 용출시키는 방법.The method of claim 14 or 15, wherein the LBTI-affinity column chromatography was washed with 50 mM sodium acetate solution (pH 5.0) containing 1 M NaCl and pH shocked with 0.2 M glycine buffer at pH 2.8 to adsorb the adsorbed protein. How to elute
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