Cloning, Expression Analysis and Enzymatic Characterization of Cathepsin L from the Inshore Hagfish (Eptatretus burgeri)

다국어 초록 (Multilingual Abstract)

Hagfish which belongs to the chordate contact cyclostomata, is important phylogenetic relationship between vertebrate and invertebrate. Cathepsins of the cysteine protease family have traditionally been thought to play a major role in intracellular protein degradation and turnover in lysosomes.
In this study, Catepsin L was cloned from Inshore hagfish (Eptatretus burgeri), the cDNA encoding ORF of the Eptatretus burgeri Cathepsin L (EbCtL) is 978 bp. The cDNA encoding proEbCtL was expressed in Escherichia coli strain BL21(DE3) using the pGEX-4T-1 expression vector system. The recombinant proEbCtL protein was overexpressed as a approximately 55 kDa fusion protein. The overproduced soluble GST-fusion protein was then applied to glutathione-Sepharose 4B column chromatography; the sample harboring the fusion protein evidenced a high degree of purity when analyzed via SDS-PAGE and Western blot analysis. Its activity was quantied by cleaving the synthetic peptide Z-FR-AMC, Z-LLE-AMC, and Suc-AAF-AMC, and the optimal pH for the protease activity was 8, 9.5, and 9, respectively.

참고문헌 (Reference)

1 Barrett, A. J., "cathepsin B, cathepsin H, and cathepsin L" 80 : 535-561, 1981

2 Guo, Y. L., "The α1/2helical backbone of the prodomains defines the intrinsic inhibitory specificity in the cathepsin L-like cysteine protease subfamily" 469 (469): 203-207, 2000

3 Petersen, T. N., "SignalP 4. 0 : Discriminating signal peptides from transmembrane regions" 8 (8): 785-786, 2011

4 Shahinian, H., "Proteomic identification of protease cleavage sites : Cell-biological and biomedical applications" 10 (10): 421-433, 2013

5 Kisselev, A. F., "Proteasome active sites allosterically regulate each other, suggesting a cyclical bite-chew mechanism for protein breakdown" 4 (4): 395-402, 1999

6 Vernet, T., "Processing of the papain precursor The ionization state of a conserved amino-acid motif within the pro region participates in the regulation of intramolecular processing" 270 (270): 10838-10846, 1995

7 Tingaud-Sequeira, A., "Phylogenetic relationships and gene expression pattern of three different cathepsin L (ctsl) isoforms in zebrafish:Ctsla is the putative yolk processing enzyme" 386 (386): 98-106, 2007

8 Dacks, J. B., "Phylogenetic and primary sequence characterization of cathepsin B cysteine proteases from the oxymonad flagellate monocercomonoides" 55 (55): 9-17, 2008

9 Kuraku, S., "Monophyly of lampreys and hagfishes supported by nuclear DNA Coded genes" 49 (49): 729-735, 1999

10 Kumar, S., "MEGA: A biologist-centric software for evolutionary analysis of DNA and protein sequences" 9 (9): 299-306, 2008

11 Turk, B., "Lysosomal cysteine proteases: More than scavengers" 1477 (1477): 98-111, 2000

12 Barrett A. J., "Hand book of Proteolytic Enzymes" Academic Press 258-, 1998

13 Wang, Z, "FV-162 is a novel, orally bioavailable, irreversible proteasome inhibitor with improved pharmacokinetics displaying preclinical efficacy with continuous daily dosing" 6 (6): e1815-, 2015

14 Zhou, J., "Evolutionary history of cathepsin L(L-like)family genes in vertebrates" 11 (11): 1016-, 2015

15 Turk, V., "Cysteine cathepsins: From structure, function and regulation to new frontiers" 1824 (1824): 68-88, 2012

16 Zavašnik Bergant, T., "Cysteine cathepsins in the immune response" 67 (67): 349-355, 2006

17 Fonović, M., "Cysteine cathepsins and their potential in clinical therapy and biomarker discovery" 8 (8): 416-426, 2014

18 Oisi, Y., "Craniofacial development of hagfishes and the evolution of vertebrates" 493 (493): 175-180, 2013

19 Z.-J. Xu, "Cloning and Characterization of the Abscisic Acid-Specific Glucosyltransferase Gene from Adzuki Bean Seedlings" American Society of Plant Biologists (ASPB) 129 (129): 1285-1295, 2002

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

21 Duncan, E. M, "Cathepsin L proteolytically processes histone H3 during mouse embryonic stem cell differentiation" 135 (135): 284-294, 2008

22 G. Song, "Cathepsin B, Cathepsin L, and Cystatin C in the Porcine Uterus and Placenta: Potential Roles in Endometrial/Placental Remodeling and in Fluid-Phase Transport of Proteins Secreted by Uterine Epithelia Across Placental Areolae" Society for the Study of Reproduction 82 (82): 854-864, 2010

23 Hall, T., "BioEdit version 7.0. 0. Distributed by the Author"

24 McDonald, J. K., "An overview of protease specificity and catalytic mechanisms: Aspects related to nomenclature and classification" 17 (17): 773-785, 1985

25 Mallatt, J., "28S and 18S rDNA sequences support the monophyly of lampreys and hagfishes" 15 (15): 1706-1718, 1998