Molecular Cloning and Functional Expression of a Fibrinolytic Protease Gene from the Polychaeta, Periserrula leucophryna

다국어 초록 (Multilingual Abstract)

Full-length cDNA encoding a fibrinolytic protease (PLFP) from the cDNA library of the polychaete,Periserrula leucophryna was cloned, sequenced and expressed in Escherichia coli. The entire cDNA of the PLFP clone was 921 bp (CDS: 41-837), including a coding nucleotide sequence of 798 bp, a 5'-untranslaed region of 40 bp, and a 3'-noncoding region of 83 bp. The ORF encoded a 265-amino acid polypeptide precursor consisting of a 36-residue signal sequence and a 229-residue mature polypeptide. The sequence alignment results of PLFP revealed sequence similarity with several fibrinolytic enzymes.
Sequence analysis revealed a conserved catalytic triad of His78, Asp126 and Ser219 residues, suggesting that PLFP is a serine protease. Mature PLFP had an apparent molecular weight of approximately 25 kDa and was produced in inclusion bodies when expressed in E. coli. Substrate specificity results that recombinant PLFP was active towards Arg-X or Lys-X and did not hydrolyze substrates with nonpolar amino acids at the P1 site. Recombinant PLFP was strongly inhibited by typical serine protease inhibitors, further indicating that PLFP is a member of the serine protease family. PLFP was able to dissolve artificial plasminogenfree fibrin, and its fibrinolytic behavior was similar to that of plasmin. Taken these results together, PLFP is a new member of the fibrinolytic enzyme family with selective specificity on fibrin, and the availability of PLFP offers an attractive alternative approach for thrombolysis therapy because rPLFP is believed to have advantages over currently used plasminogen activators, that is, lower price and lower side effect.

참고문헌 (Reference)

1 Sinnaeve, P., "Thrombolytic therapy. State of the art" 103 : 71-79, 2001

2 Sazonova, I. Y., "The mechanism of a bacterial plasminogen activator intermediate between streptokinase and staphylokinase" 276 : 12609-12613, 2001

3 Sugimoto, S., "The fibrinolytic activity of a novel protease derived from a tempeh producing fungus, Fusarium sp. BLB" 71 : 2184-2189, 2007

4 Astrup, T., "The fibrin plate method for the estimation of fibrinolytic activity" 40 : 346-351, 1952

5 Wardlaw, J. M., "Systematic review of evidence on thrombolytic therapy for acute ischaemic stroke" 350 : 607-614, 1997

6 Joo, H. S., "Simple methods for alkaline protease purification from the polychaeta, Periserrula leucophryna" 37 : 299-303, 2001

7 Wang, F., "Purification, characterization and crystallization of a group of earthworm fibrinolytic enzymes from Eisenia fetida" 25 : 1105-1109, 2003

8 You, W. K., "Purification and molecular cloning of a novel serine protease from the centipede, Scolopendra subspinipes mutilans" Pergamon Press Ltd. 34 (34): 239-250, 2004

9 Matsubara, K., "Purification and characterization of two fibrinolytic enzymes from a marine green alga, Codium intricatum" 119 : 177-181, 1998

10 Yang, J. S., "Purification and characterization of an SDS-activated fibrinolytic enzymes from Eisenia fetida" 118 : 623-631, 1997

11 Dametto, M., "Purification and characterization of a trypsin-like enzyme with fibrinolytic activity present in the abdomen of horn fly, Haematobia irritans irritans (Diptera: Muscidae)" 19 : 515-521, 2000

12 Ahn, M. Y., "Purification and characterization of a serine protease with fibrinolytic activity from the dung beetles, Catharsius molossus" PERGAMON-ELSEVIER SCIENCE LTD 112 (112): 339-347, 2003

13 Hahn, B. S., "Purification and characterization of a plasmin-like protease from Tenodera sinensis (Chinese mantis)" 31 : 573-581, 2001

14 Hua, Y., "Purification and characterization of a novel fibrinolytic enzyme from Bacillus sp. nov. SK006 isolated from an Asian traditional fermented shrimp paste" 56 : 1451-1457, 2008

15 Matsubara, K., "Purification and characterization of a fibrinolytic enzyme and identification of fibrinogen clotting enzyme in a marine green alga, Codium divaricatum" 125 : 137-143, 2000

16 LEE, JONG-SUK,, "Purification and Characterization of Two Novel Fibrinolytic Proteases fromMushroom, Fomitella fraxinea" 한국미생물·생명공학회 16 (16): 264-271, 2006

17 Ali, U. F., "Production and some properties of fibrinolytic enzyme from Rhizomucor miehei (Cooney & Emerson) Schipper" 4 : 892-899, 2008

18 Baker, B. J., "Nucleotide sequence encoding the snake venom fibrinolytic enzyme atroxase obtained from a Crotalus atrox venom gland cDNA library" 317 : 357-364, 1995

19 Toombs, C. F., "New directions in thrombolytic therapy" 1 : 164-168, 2001

20 이형환, "Molecular Cloning, Sequencing, and Expression of a Fibrinolytic Serine-protease Gene from the Earthworm Lumbricus rubellus" 한국생화학분자생물학회 37 (37): 574-581, 2004

21 Kim, J. B., "Identification of a fibrinolytic enzyme by Bacillus vallismortis and its potential as a bacteriolytic enzyme against Streptococcus mutans" 29 : 605-610, 2007

22 Kwang Bon Koo, "Fibrinolytic Activity of a Novel Serine Protease from the Hemolymph of a Polychaeta, Periserrula leucophryna" 한국응용생명화학회 53 (53): 149-157, 2010

23 Nakajima, N., "Earthwormserine protease: characterization, molecular cloning, and application of the catalytic functions" 23 : 191-212, 2003

24 Kho, C. W., "Confirmation of Vpr as a fbrinolytic enzyme present in extracellular proteins of Bacillus subtilis" 39 : 1-7, 2005

25 Choi, N. S., "Cloning, expression, and fibrin(ogen)olytic properties of a subtilisin DJ-4 gene from Bacillus sp. DJ-4" 236 : 325-331, 2004

26 Peng, Y., "Cloning and expression of a fibrinolytic enzyme (subtilisin DFE) gene from Bacillus amyloliquefaciens DC-4 in Bacillus subtilis" 155 : 167-173, 2004

27 Hyeon-Deok Jo, "Cloning and Overexpression of aprE3-17 Encoding the Major Fibrinolytic Protease of Bacillus licheniformis CH 3-17" 한국생물공학회 16 (16): 352-359, 2011

28 Han Seung Joo, "Cloning and Expression of the Cathepsin F-like Cysteine Protease Gene in Escherichia coli and Its Characterization" 한국미생물학회 45 (45): 158-167, 2007

29 Laemmli, U. K, "Cleavage of structural proteins during the assembly of the head of bacteriophage T4" 227 : 680-685, 1970

30 Jang Won Choi, "Characterization, Cloning and Expression of the Ferritin Gene fromthe Korean Polychaete, Periserrula leucophryna" 한국미생물학회 44 (44): 54-63, 2006

31 Hee Gon Kim, "Characterization of Methylophaga sp. strain SK1 Cytochrome cL Expressed in Escherichia coli" 한국미생물학회 43 (43): 499-502, 2005

32 Wang, P., "Catalytic and fibrinolytic properties of recombinant urokinase plasminogen activator from E. coli, mammalian, and yeast cells" 100 : 461-467, 2000

33 Siigur, J., "Biochemical characterization of lebetase, a direct–acting fibrinolytic enzyme from Vipera lebetina snake venom" 90 : 39-49, 1998

34 Chung, K. H., "Biochemical and biological properties of a fibrinolytic enzyme from Korean snake (Agkistrodon halys) venom" 26 : 370-377, 1993

35 Rym, A., "BSF1 fibrinolytic enzyme from a marine bacterium Bacillus subtilis A26: Purification, biochemical and molecular characterization" 44 : 1252-1259, 2009

36 Rayda Siala, "Arthrobacter arilaitensis Re117 Oxidant-stable Alkaline Metalloprotease: Purification and Biochemical Characterization" 한국생물공학회 17 (17): 556-564, 2012

37 Randolph, A., "Amino acid sequence of fibrolase, a direct-acting fibrinolytic enzyme from Agkistrodon contortrix contortrix venom" 1 : 590-600, 1992

38 Anissa Haddar, "Alkaline Proteases Produced by Bacillus licheniformis RP1 Grown on Shrimp Wastes: Application in Chitin Extraction, Chicken Featherdegradation and as a Dehairing Agent" 한국생물공학회 16 (16): 669-678, 2011

39 Bradford, M. M., "A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding" 72 : 248-254, 1976

40 Zhang, Y., "A novel fibrinolytic serine protease from the polychaete Nereis (Neanthes) virens (Sars): Purification and characterization" 89 : 93-103, 2007

41 Simkhada, J. R., "A novel fibrinolytic protease from Streptomyces sp. CS684" 45 : 88-93, 2010

42 Tabor, S., "A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes" 82 : 1074-1078, 1985