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Tytuł pozycji:

Prokaryotic expression, evaluation, and prediction of the structure and function of the ecarin metalloproteinase domain.

Tytuł:
Prokaryotic expression, evaluation, and prediction of the structure and function of the ecarin metalloproteinase domain.
Autorzy:
Mohammadi N; Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran.
Bandehpour M; Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran.; Department of Biotechnology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Sotoodehnejadnematalahi F; Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran.
Kazemi B; Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran.; Department of Biotechnology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Źródło:
Proteins [Proteins] 2022 Mar; Vol. 90 (3), pp. 802-809. Date of Electronic Publication: 2021 Nov 17.
Typ publikacji:
Journal Article; Research Support, Non-U.S. Gov't
Język:
English
Imprint Name(s):
Publication: New York, NY : Wiley-Liss
Original Publication: New York : Alan R. Liss, c1986-
MeSH Terms:
Endopeptidases/*chemistry
Metalloproteases/*chemistry
Recombinant Proteins/*chemistry
Animals ; Endopeptidases/pharmacology ; Escherichia coli/enzymology ; Escherichia coli/genetics ; Humans ; Metalloproteases/genetics ; Metalloproteases/metabolism ; Recombinant Proteins/genetics ; Recombinant Proteins/metabolism ; Thrombin/chemistry ; Thrombosis/drug therapy ; Viperidae
References:
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Yonemura H, Imamura T, Nakatake H, Soejima K, Nozaki C, inventors; Chemo Sero Therapeutic Research Institute Kaketsuken, assignee. Genetically modified ecarin and process for producing the same. United States patent application US 10/482,925. 2005.
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Contributed Indexing:
Keywords: I-TASSER; ecarin; metalloproteinase domain; recombinant protein
Substance Nomenclature:
0 (Recombinant Proteins)
EC 3.4.- (Endopeptidases)
EC 3.4.- (Metalloproteases)
EC 3.4.21.5 (Thrombin)
EC 3.4.24.- (ecarin)
Entry Date(s):
Date Created: 20211105 Date Completed: 20220316 Latest Revision: 20220316
Update Code:
20240105
DOI:
10.1002/prot.26275
PMID:
34739152
Czasopismo naukowe
Ecarin is one of the most widely used drug compounds in blood clotting experiments and is used to monitor and treat many diseases such as cancer, liver, lupus, and cardiovascular disease. The metalloproteinase domain is known as the active site of ecarin. In this study, an ecarin metalloproteinase cassette was designed and synthesized in the pUC57 vector. The gene fragment was released and cloned into the pET-28a vector and expressed in Escherichia coli. The recombinant protein was confirmed by western blotting. Enzyme activity was estimated by a laboratory coagulation test, and prothrombin time and tertiary structure were determined by using the Iterative Threading ASSEmbly Refinement (I-TASSER) server. Data from blood clotting tests for the produced ecarin activity were analyzed using an independent t test. As per I-TASSER server prediction, model 1 with the highest confidence score 0.95, template modeling score (0.84 ± 0.08), and root mean square deviation (3.5 ± 2.4 Å) was considered as the best model, and the 2e3xA enzyme was more similar to the target protein. The predictive results helped to better understand the relationship between the structure and function of the ecarin metalloproteinase domain. Also, the production of this active site in the prokaryotic expression system, which is simpler and more cost-effective than the production of the eukaryotic system, showed that this recombinant ecarin could be used as a substitute for the raw snake venom of Echis carinatus because it converts prothrombin into thrombin, and its activity, as estimated using the prothrombin time test, was found to be faster than normal ecarin.
(© 2021 Wiley Periodicals LLC.)

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