4.1 Article

Direct Cloning, Expression and Purification of Human Activated Thrombin in Prokaryotic System and CD Analysis Report of Produced Thrombin: Molecular Characterization of Recombinant Thrombin

Journal

Publisher

SPRINGER
DOI: 10.1007/s10989-020-10046-2

Keywords

Thrombin; Escherichia coli; Expression; Purification; Dialysis; Circular Dichroism

Funding

  1. School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences

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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 x his-tag was expressed in pET-28a(+)-Escherichia coliOrigami 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 inE. 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.

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