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Molecular Cloning, Expression and Purification of Fibrinolytic Enzyme from Medicinal Mushroom, Cordyceps militaris

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Author(s)
김재성
Issued Date
2006
Abstract
Blood clots are formed by the conversion of fibrinogen into fibrin via the proteolytic action of thrombin and subsequently, the formation of insoluble fibrin clots. These fibrin clots are dissolved by the hydrolysis of plasmin, which is activated from plasminogen by tissue plasmino gen activator (PA). The hydrolysis of fibrin is also known as fibrinolysis. Fibrin clot formation and fibrinolysis are normally well balanced in biological systems. However, when fibrin is not hydrolyzed due to some disorder, thromboses can occur, leading to serious consequences in human body such as apopelexy, arterio sclerosis, cerebral infraction, cerebral hemorrhage and myocardial infarction.
The fibrinolytic agents available today for clinical use are mostly plasmin ogen activator such as a tissue-type plasminogen activator (tPA), a urokinase -type plasminogen activator (uPA), and the bacterial plasminogen activator streptokinase (SK). Despite their widespread use, all these agents have unde sired side effects, exhibit low specificity for fibrin, and are also relatively expensive. Therefore, the searches for other fibrinolytic enzymes from vario us sources are being continued. Recently, an investigation was conducted involving the isolation of fibrinolytic enzyme from natural extracts, as the fibrinolytic proteases used in thrombolytic therapy exhibit high specificity for fibrin, and are relatively inexpensive.
Several nontoxic mushrooms contain biologically active substances. There extracts have been reported to exert hematological, antiviral, anti tumorigenic, hypotensive, and hepatoprotective effects. Mushrooms constitute an important source of thrombolytic agents. Korean traditional anecdotes suggest that mushrooms can be, and have been, used in the treatment and prevention of thrombosis. Furthermore, some reports have described fibrinolytic activity occurring in some edible mushrooms, including Flammulina velutipes, Pleurotus ostreatus, Grifola frondosa, Tricholoma saponaceum, C. militaris and Armillaria mellea.
A fibrinolytic enzyme was purified from the C. militaris by ion-exchange chromatography followed by gel filtration and fast protein liquid chromato graphy. The purification protocol resulted in a 191.8-fold purification of the enzyme, with a final yield of 12.9%. The apparent molecular mass of the purified enzyme was estimated to be 52 kDa by SDS-PAGE, fibrin-zymo graphy and gel filtration chromatography, which revealed a monomeric form of the enzyme. The optimal reaction pH and temperature were pH 7.4, and 37 °C, respectively. This protease effectively hydrolyzed fibrinogen, preferentially digesting the Aα-chain over the Bβ- and γ-chains. Enzyme activity was inhibited by Cu2+ and Co2+, but enhanced by the addition of Ca2+ and Mg2+ ions. Furthermore, fibrinolytic enzyme activity was potently inhibited by phenylmethylsulfonyl fluoride (PMSF) and 4-amidinophenyl-methane sulfonyl fluoride (APMSF), and was found to exhibit a higher specificity for the substrate S-2586 for chymotrypsin, indicating that the enzyme is a chymotrypsin-like serine protease. The first 19 amino acid residues of the N-terminal sequence were ALTTQSN VTHGLATISLRQ, which is extremely similar to the subtilisin-like serine protease PR1J (NCBI Accession No. CAC95048).
The subtilisin like fibrinolytic enzyme gene of C. militaris was cloned and its nucleotide sequence was determined. The nucleotide sequence revealed one large open reading frame, composed of 1,197 base pairs which were translated into 398 amino acids. The deduce amino acid sequence of fibrinolytic enzyme from C. militaris is composed of two domains peptidase -S8 and subtilisin-N domain. Peptidase-S8 (Thr139~Asn395) belongs to subtil ase family which is a family of serine proteases. They appear to have indep endently and convergently evolved an Asp/Ser/His catalytic triad, like that found in the trypsin serine proteases. Subtilisin-N domain (Asp33~His116) has subtilisin N-terminal region. This family is found at the N-terminus of a number of subtilisins.
The Cmfe 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 Cmfe was inserted and orientation in the plasmid pQE+ Cmfe was confirmed by double restriction enzyme mapping. The plasmid pQE30+Cmfe were transformed into E. coli expression host M15. All the picked clones were expressed the CmFE with a predicted band around 52 kDa. Interestingly all the recombinant protein was found in insoluble pellete which were conformed by the western blot analysis.
In conclusion, the fibrinolytic enzyme purified from C. militaris exhibits a profound fibrinolytic activity, and also evidences relatively high substrate specificity to fibrin. Therefore, C. militaris 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 provided a way to obtain a single component with fibrinolytic activity and baseline information for further study on understanding of the structure and function relationship of the enzymes.
Alternative Title
밀리타리스 동충하초로부터 혈전분해효소 정제 및 특성
Alternative Author(s)
Kim, Jae-Sung
Affiliation
조선대학교 대학원
Department
일반대학원 유전자과학과
Advisor
김성준
Awarded Date
2006-02
Table Of 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
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-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
IV. DISCUSSION = 70
V. REFERENCE = 76
Degree
Doctor
Publisher
조선대학교 대학원
Citation
김재성. (2006). Molecular Cloning, Expression and Purification of Fibrinolytic Enzyme from Medicinal Mushroom, Cordyceps militaris.
Type
Dissertation
URI
https://oak.chosun.ac.kr/handle/2020.oak/6057
http://chosun.dcollection.net/common/orgView/200000232713
Appears in Collections:
General Graduate School > 4. Theses(Ph.D)
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