Split intein-mediated backbone cyclization enhances the stability and activity of staphylokinase, a potent fibrin-selective plasminogen activator.

Autor: Baharian A; Biochemistry Research Group, Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada., Ishida H; Biochemistry Research Group, Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada., Sillner C; Biochemistry Research Group, Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada., Vogel HJ; Biochemistry Research Group, Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada. Electronic address: vogel@ucalgary.ca.
Jazyk: angličtina
Zdroj: International journal of biological macromolecules [Int J Biol Macromol] 2024 Aug; Vol. 275 (Pt 1), pp. 133448. Date of Electronic Publication: 2024 Jun 28.
DOI: 10.1016/j.ijbiomac.2024.133448
Abstrakt: Staphylokinase (Sak), a small 15 kDa globular protein that is secreted by certain strains of Staphylococcus aureus, shows a potent fibrin-selective thrombolytic activity. Earlier work has shown that Sak could potentially become a low-cost alternative to currently used thrombolytic agents, such as tissue plasminogen activator (tPA). In attempts to improve its potential for clinical applications, numerous modifications of Sak have already been investigated. Here, we have characterized a novel Sak modification, cyclized Sak (cyc-Sak), which was prepared through split-intein mediated protein backbone cyclization. We have characterized the structure, stability and the activity of cyc-Sak using biophysical techniques, limited proteolysis studies and plasminogen (PG)-activation assays. Our results show that cyc-Sak possesses an identical structure, enhanced stability, resistance to proteolysis by exoproteases and improved PG-activation properties compared to its linear counterpart. It can be over-expressed with high yield in the cytoplasm of Escherichia coli and is easily purified in a two-step process. The intein-mediated cyclization occurs spontaneously in vivo during protein expression and does not necessitate further modification steps after purification of the protein. Furthermore, covalent Sak cyclization could be readily combined with other Sak modifications previously proposed, to generate an effective thrombolytic agent with lower immunogenicity and improved stability and activity.
Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
(Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)
Databáze: MEDLINE
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