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ABSTRACT Molecular Cloning, Expression And 20062006年度年度 2 2月月 博士學位論文 MMMolecularMolecular CloningCloning,, Expression and PPPurificationPurification of Fibrinolytic Enzyme from Medicinal MMMushroom,Mushroom, Cordyceps mmmilitarismilitaris 朝 鮮 大 學 校 大 學 院 遺 傳 子 科 學 科 金 在 城 MMMolecularMolecular CloningCloning,, Expression and PPPurificationPurification of Fibrinolytic EnzymEnzymee from Medicinal MMMushroom,Mushroom, Cordyceps militaris 밀리타리스 동충하초로부터 혈전분해효소 정제 및 특성 분석과 유전자 크로닝 20052005年年 月 日 朝 鮮 大 學 校 大 學 院 遺 傳 子 科 學 科 金 在 城 MMMolecularMolecular CloningCloning,, Expression and PPPurificationPurification of Fibrinolytic Enzyme from Medicinal MMMushroom,Mushroom, CorCordycepsdyceps militaris 指導敎授 金 成 俊 이 論文을 理學博士學位 申請論文으로 제출함 20052005年年 月 日 朝 鮮 大 學 校 大 學 院 遺 傳 子 科 學 科 金 在 城 金在城의 博士學位論文을 認准함... 委員長 朝鮮大學校 敎授 印 委 員 朝鮮大學校 敎授 印 委 員 益 山 大 學 敎授 印 委 員 東新大學校 敎授 印 委 員 朝鮮大學校 敎授 印 20052005年年 月 日 朝 鮮 大 學 校 大 學 院 CONTENTS LIST OF TABLES............................................................................v LIST OF FIGURES ........................................................................ vi ABBREVIATIONS ..........................................................................x ABSTRACT .....................................................................................xii I. INTRODUCTION ...................................................................................1 II. MATERIALS AND METHODS ......................................................14 II-A. Materials ................................................................................................14 II-B. Cultivation of C. militaris ......................................................................15 II-C. Preparation of crude extracts ..............................................................16 II-D. Assay of fibrinolytic activity. ...............................................................17 II-D-1. Assay of proteolytic activity ............................................................17 II-D-2. The preparation of fibrin plate .......................................................17 II-E. Purification of fibrinolytic enzyme ......................................................18 II-E-1. Determination of protein concentration ........................................18 II-E-2. Cation exchange chromatography on CM-cellulose column .......18 II-E-3. Anion exchange chromatography on DEAE sephadex A-50 column ..............................................................................................18 i II-E-4. Gel filtration chromatography on sephadex G-75 column ..........19 II-E-5. Fast protein liquid chromatography (FPLC) on HiLoad 16/60 Superdex 75 pg column ..................................................................19 II-F. Molecular weight determination ..........................................................20 II-F-1. Size exclusion using FPLC ..............................................................20 II-F-2. SDS-PAGE ........................................................................................20 II-F-3. Fibrin-zymography ..........................................................................21 II-G. Determination of N-terminal sequence and structure alignment .....21 II-H. Characterization of purified fibrinolytic enzyme ..............................22 II-H-1. Degradation patterns of fibrin and fibrinogen .............................22 II-H-2. Effect of temperature and pH on enzyme activity ........................22 II-H-3. Effect of metal ions and protease inhibitors on the enzyme activity .............................................................................................23 II-H-4. Amidolytic activity of the enzyme ..................................................23 II-I. The molecular cloning of fibrinolytic enzyme gene .............................25 II-I-1. Total RNA isolation ..........................................................................25 II-I-2. Reverse transcriptase-polymerase chain reaction (RT-PCR) .......26 II-I-3. 5′ and 3 ′ rapid amplification of cDNA ends (RACE) .....................26 II-I-4. DNA sequencing and analysis ..........................................................30 II-I-5. The cloning of Cmfe ..........................................................................30 II-I-6. Northern hybridization .....................................................................32 ii II-J. The expression of fibrinolytic enzyme gene .........................................32 II-J-1. Construction of expression vector for recombinant fibrinolytic enzyme gene ....................................................................................32 II-J-2. Expression and purif ication of recombinant fibrinolytic enzyme .33 II-J-3. Western blot assay ............................................................................35 III-C. RESULTS ..........................................................................................36 III-A. Purification of fibrinolytic enzyme ....................................................36 III-B. Fibrinolytic activity assay ...................................................................36 III-C. Determination of molecular weight ...................................................42 III-D. N-terminal amino acid sequence of fibrinolytic enzyme from C. militaris ...............................................................................................42 III-E. Effect of pH and temperature on fibrinolytic activity ......................46 III-F. Effect of inhibitors and metal ions on the fibrinolytic activity ........46 III-G. Analysis of fibrinolysis and fibrinogenolysis .....................................50 III-H. Amidolytic activity of fibrinolytic enzyme from C. militaris ...........56 III-I. 3’-RACE and 5’-RACE for molecular cloning of Cmfe ....................56 III-J. Molecular cloning of Cmfe ...................................................................59 III-K. Analysis of deduce amino acid sequence of CmFE ..........................67 III-L. Gene expression of Cmfe cDNA ..........................................................67 iii IV. DISCUSSION ........................................................................................70 V. REFERENCE ..........................................................................................76 감사의 글..........................................................................................................89 iv LIST OF TABLES Table 1. List of synthertic Chromogenic substrate...................................24 Table 2. Oligodeoxynucleotide primers used for synthesis and amplify cation of partial, 3 ′-RACE and 5 ′-RACE of the Cmfe cDNA..27 Table 3. Purified fibrinolytic enzyme activity yields from C. militaris. ..41 Table 4. Effect of various protease inhibitors on the activity of the purified fibrinolytic enzyme from C. militaris. ...........................................51 Table 5. Effect of metal ions on the activity of the fibrinolytic enzyme from C. militaris. ...........................................................................52 v LIST OF FIGURES Fig. 1. Schematic diagram of blood clotting ................................................2 Fig. 2. The pathway of blood clotting by intrinsic and extrinsic path way. .............................................................................................................3 Fig. 3. Schematic diagram of fibrinolytic system. ......................................4 Fig. 4. Hemostasis between fibrinolysis and thrombosis. ..........................5 Fig. 5. The strategy of 5’RACE amplification of unknown sequences at the 5’ end of the mRNA...................................................................28 Fig. 6. The strategy of 3’RACE amplification of unknown sequences at the 3’ end of the mRNA...................................................................29 Fig. 7. Construction of Cmef cDNA with pGEM T-easy vector...............31 Fig. 8. Construction of expression vector for recombinant fibrinolytic enzyme gene......................................................................................34 Fig. 9. Purification of fibrinolytic enzyme from C. militaris using cation- exchange chromatography on CM-cellulose column. ..................37 Fig. 10. Purification of fibrinolytic enzyme from C. militaris using anion- exchange chromatography on DEAE sephadex A-50 column. 38 vi Fig. 11. Purification of fibrinolytic enzyme from C. militaris using gel filtration on sephadex G-75 column...........................................39 Fig. 12. Purification of fibrinolytic enzyme from C. militaris using fast protein liquid chromatography (FPLC) on HiLoad 16/60 Supe rdex 75 pg column........................................................................40 Fig. 13. Fibrin plate assay of purified fibrinolytic enzyme from C. militaris ..........................................................................................43 Fig. 14. Molecular weight determination of fibrinolytic enzyme from C. militaris using size-exclusion chromatography on HiLoad 16/60 Superdex 75 pg column. ...............................................................44 Fig. 15. Molecular weight determination of fibrinolytic enzyme from C. militaris using SDS-PAGE and fibrin zymography..................45 Fig. 16. An aligment of the amino-terminal sequence of purified fibrinolytic enzyme from C. militaris with those of the serine protease family from Metarhizium anisopliae . ........................................................47 Fig. 17. Effects of various pH on the activity
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