Direct Cloning, Expression and Purification of Human Activated Thrombin in Prokaryotic System and CD Analysis Report of
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Direct Cloning, Expression and Purification of Human Activated Thrombin in Prokaryotic System and CD Analysis Report of Produced Thrombin: Molecular Characterization of Recombinant Thrombin Amin Barkhordari1 · Abbas Behzad‑Behbahani2 · Vahid Jajarmi1,3 · Mojgan Bandehpour3 · Gholamreza Rafiei‑Dehbidi2 · Fatemeh Safari2 · Fereydoun Mahboudi4 · Bahram Kazemi1,3 Accepted: 5 February 2020 © Springer Nature B.V. 2020
Abstract Thrombin is a serine protease with a key role in coagulation and controlling hemostasis. Versatile usage of thrombin in surgeries as well as, its multi-functional role in hemostasis has encouraged scientists to produce recombinant human thrombin. Since, the generation of recombinant thrombin in eukaryotic host cells is so expensive and time-consuming. Hence, in this study, the prokaryotic workhorse was applied for direct production of activated-thrombin to achieve a cost and time efficient method. In this study, the construction consist of light-heavy chain of human thrombin fused with a 6 × his-tag was expressed in pET-28a(+)-Escherichia coli Origami type 2 system. Ni2+-NTA affinity chromatography was used for purification of recombinant thrombin. Then purified protein was characterized by SDS-PAGE analysis, western and DOT blotting. For proper refolding, the purified protein was undergone air oxidation. Circular Dichroism was performed to evaluate the proper folding and secondary structure of protein. Consequently, bioactivity assay was carried out to analyze the proteolytic activity of recombinant thrombin, which showed the formation of clot by conversion of fibrinogen to fibrin. Production of high yield activated recombinant thrombin in E. coli, was more cost and time-effective, than in eukaryotic systems. At the end, we report the expression and purification method used in this article provides a simple, rapid and cost-effective procedure to produce activated thrombin. Keywords Thrombin · Escherichia coli · Expression · Purification · Dialysis · Circular Dichroism
Introduction Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10989-020-10046-2) contains supplementary material, which is available to authorized users. * Mojgan Bandehpour [email protected] * Bahram Kazemi [email protected] 1
Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
2
Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran
3
Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran
4
Biotechnology Research Center, Pasteur Institute of Iran, 1316943551 Tehran, Iran
Thrombin (Factor IIa or fibrinogenase) as an endolytic serine protease plays a key role in blood clotting cascade (Andrew et al. 1987). Thrombin triggers a broad range of physiological activities by cleavage of various plasma proteins or thrombin receptors on
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