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느타리버섯으로부터 혈전분해효소 정제 및 특성분석과 유전자 발현

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Author(s)
심명화
Issued Date
2006
Abstract
Fibrinolytic enzymes dissolve the blood clots, which are formed by the conversion of fibrinogen into fibrin via the proteolytic action of thrombin. When clots are not lysed, they accumulate in blood vessels and cause thrombosis leading to myocardial infarction and other cardiovascular diseases. The major thrombolytic agents are classified into two types. The plasminogen activators, such as urokinase, tPA (tissue type plasminogen activator), and streptokinase, which activate plasminogen to plasmin, and the plasmin-like proteins, such as nattokinase and lumbrokinase, which can directly degrade the fibrin.
Mushrooms constitute an important source of thrombolytic agents. Many fibrinolytic enzymes were identified in fruiting body of different medicinal mushrooms, such as Armillaria mellea metalloprotease (AMMP) from Armillaria mellea, Grifora frondosa aminopeptidase(GfAP) from Grifora frondosa and Pleurotus ostreatus metalloprotease (PoMTP) from Pleurotus ostreatus. All of these enzymes are Zn-metalloprotease group proteins and belong to the same wood rotting fungi. Indeed the presence of fibrinolytic enzymes in fruiting body of some mushrooms have been identified although the presence of those enzymes in their cultured mycelia is not clear. Therefore we tried to isolate the fibrinolytic enzymes from cultured mycelia of mushroom. The purified enzyme was designated as Pleurotus ostreatus fibrinolytic enzyme (PoFE).
Fibrinolytic enzyme was purified from the edible mushroom, Pleurotus ostreatus by ion-exchange chromatography followed by gel filtration and fast protein liquid chromatography (FPLC). The purification protocol resulted in an 877-fold purification of the enzyme, with a final yield of 6.5%. The apparent molecular mass of the purified enzyme was estimated to be 32 kDa by SDS-PAGE, fibrin-zymography and gel filtration chromatography, which revealed a monomeric form of the enzyme. The optimal reaction pH and temperature were pH 6.5, and 35 °C, respectively. This protease effectively hydrolyzed fibrinogen, preferentially digesting the Aα-chain and the Bβ-chain over γ-chains. Enzyme activity was enhanced by the addition of Ca2+, Zn2+ and Mg2+ ions. Furthermore, fibrinolytic enzyme activity was potently inhibited by tetrasodium ethylenediaminetetraacetate (EDTA), and it was found to exhibit a higher specificity for the substrate S-2586 for chymotrypsin, indicating that the enzyme is a chymotrypsin-like metalloprotease. The first 19 amino acid residues of the N-terminal sequence were ALRKGGAAALNIYSVGFTS, which is extremely similar to the Pleurotus ostreatus metalloprotease purified from fruiting body of Pleurotus ostreatus (NCBI Accession No. AAU94648.1). The gene encoding a fibrinolytic enzyme, PoFE that belongs to the metalloprotease family was cloned and expressed in Escherichia coli. The gene has 867 base pairs and encodes an active protein of 288 amino acids. Sequence analysis suggests that the fibrinolytic enzyme from Pleurotus ostreatus consists of a Zn-binding domain which is amino acid sequence HEXXHXUGUXH (His198~His208). The PoFE was cloned to plasmid pQE30 in the correct reading frame, with an N-terminal 6X His-tag sequence under the control of the T5 promoter and lac operator. The PoFE was inserted and orientation in the plasmid pQE30-pofe was confirmed by double restriction enzyme mapping. The plasmid pQE30-pofe were transformed into Escherichia coli (E. coli) expression host M15 (pREP4). All the picked clones were expressed the PoFE with a predicted band around 32 kDa. It is interesting to note that all of the recombinant protein was found in inclusion bodies which were confirmed by the western blot analysis.
In conclusion, the fibrinolytic enzyme purified from Pleurotus ostreatus exhibits a profound fibrinolytic activity, and also evidences relatively high substrate specificity to fibrin. Therefore, Pleurotus ostreatus may become a new source for thrombolytic agents, and can be used to develop therapeutic agents for the treatment of thrombosis. In addition, the work described here provides a way to obtain a single component with fibrinolytic activity and a baseline information for further study on understanding of the structure-function relationship of the enzymes.
Alternative Title
Molecular olecular Cloning, Expression and Purification of Fibrinolytic Enzyme from Pleurotus ostreatus
Alternative Author(s)
Shen, Ming-Hua
Affiliation
조선대학교 대학원
Department
일반대학원 유전자과학과
Advisor
김성준
Awarded Date
2006-02
Table Of Contents
LIST OF TABLES = Ⅴ
LIST OF FIGURES = Ⅵ
ABBREVIATIONS = Ⅸ
ABSTRACT = XI
Ⅰ 서론 = 1
Ⅱ 재료 및 방법 = 9
Ⅱ-1 재료 = 9
Ⅱ-1-1 균주 = 9
Ⅱ-1-2 시약 = 9
Ⅱ-1-3 느타리버섯 균사체 배양 = 10
Ⅱ-2 혈전분해효소의 분리정제 = 10
Ⅱ-2-1 조추출물 조제 = 10
Ⅱ-2-2 단백질분해활성 및 혈전분해활성 검색 = 11
Ⅱ-2-3 조단백질 분리 = 11
Ⅱ-2-4 단백질 정량 = 12
Ⅱ-2-5 단백질분해활성 측정 = 12
Ⅱ-2-6 Cation-exchange chromatography = 12
Ⅱ-2-7 Anion-exchange chromatography = 12
Ⅱ-2-8 Gel-filtration chromatography = 13
Ⅱ-2- Fast protein liquid chromatography = 13
Ⅱ-2-10 혈전분해효소의 분리 = 13
Ⅱ-3 분자량 측정 = 13
Ⅱ-3-1 FPLC를 이용한 분자량 측정(size exclusion) = 14
Ⅱ-3-2 SDS-PAGE를 이용한 분자량 측정 = 14
Ⅱ-3-3 SDS-fibrin zymography를 이용한 분자량 측정 = 14
Ⅱ-4 N-말단 아미노산 서열분석 = 15
Ⅱ-5 정제효소의 특성 분석 = 15
Ⅱ-5-1 정제효소의 기질특이성 분석 = 15
Ⅱ-5-2 효소활성에 대한 금속이온 및 단백질분해효소 억제제의 영향 = 16
Ⅱ-5-3 효소활성에 대한 pH의 영향 = 16
Ⅱ-5-4 효소활성에 대한 온도의 영향 = 18
Ⅱ-5-5 Fibrin 및 Fibrinogen에 대한 분해특성 분석 = 18
Ⅱ-6 Total RNA isolation = 18
Ⅱ-7 혈전분해효소 유전자에 대한 특이적 primer의 제작 = 19
Ⅱ-8 RT-PCR = 19
Ⅱ-9 Cloning을 위한 형질전환 = 20
Ⅱ-10 DNA 염기서열 분석 = 20
Ⅱ-11 Construction of expression vector for recombinant fibrinolytic enzyme gene = 21
Ⅱ-12 Expression and purification of fibrinolytic enzyme = 22
Ⅱ-13 SDS-PAGE = 25
Ⅱ-14 Western blot analysis = 25
Ⅲ 결과 = 26
Ⅲ-1 단백질분해활성 및 혈전분해활성 = 26
Ⅲ-2 느타리버섯 균사체로부터 혈전분해효소의 정제 = 26
Ⅲ-2-1 조단백질 분리 = 26
Ⅲ-2-2 혈전분해효소의 정제 = 28
Ⅲ-3 혈전분해효소의 분자량 측정 = 28
Ⅲ-3-1 FPLC 를 이용한 분자량 측정(size exclusion) = 28
Ⅲ-3-2 SDS-PAGE 및 fibrin zymography를 이용한 분자량 측정 = 29
Ⅲ-4 혈전분해효소의 N-말단 아미노산 서열분석 = 29
Ⅲ-5 혈전분해효소의 특성분석 = 38
Ⅲ-5-1 기질특이성 분석 = 38
Ⅲ-5-2 효소활성에 대한 금속이온의 영향 = 38
Ⅲ-5-3 효소활성에 대한 Protease inhibitor의 영향 = 38
Ⅲ-5-4 효소활성에 대한 pH의 영향 = 39
Ⅲ-5-5 효소활성에 대한 온도의 영향 = 39
Ⅲ-5-6 Fibrinolysis와 Fibrinogenolysis pattern 분석 = 39
Ⅲ-5-6-1 Fibrinogenolysis = 39
Ⅲ-5-6-2 Fibrinolysis = 45
Ⅲ-6 Cloning and sequence analysis of pofe = 45
Ⅲ-7 Analysis of deduce amino acid sequence of PoFE = 48
Ⅲ-8 Gene expression of pofe = 49
Ⅳ 고찰 = 62
Ⅴ 적요 = 67
Ⅵ 참고문헌 = 69
Degree
Doctor
Publisher
조선대학교 대학원
Citation
심명화. (2006). 느타리버섯으로부터 혈전분해효소 정제 및 특성분석과 유전자 발현.
Type
Dissertation
URI
https://oak.chosun.ac.kr/handle/2020.oak/6130
http://chosun.dcollection.net/common/orgView/200000232869
Appears in Collections:
General Graduate School > 4. Theses(Ph.D)
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