Improving the therapeutic potential of staphylokinase, a potent thrombolytic agent

Baharian, Azin

Improving the therapeutic potential of staphylokinase, a potent thrombolytic agent

dc.contributor.advisor	Vogel, Hans J.
dc.contributor.author	Baharian, Azin
dc.contributor.committeemember	MacDonald, Justin A.
dc.contributor.committeemember	MacCallum, Justin
dc.date	2023-11
dc.date.accessioned	2023-05-30T16:30:31Z
dc.date.available	2023-05-30T16:30:31Z
dc.date.issued	2023-05-26
dc.description.abstract	Staphylokinase (Sak) is a small bacterial-derived protein (15.5 kDa) that can hydrolyze fibrin-rich blood clots by activating the conversion of plasminogen into plasmin. Several studies have shown the effectiveness of Sak as a thrombolytic agent that could be used to treat stroke or cardiac arrest. However, its short half-life in blood circulation and immunogenicity are the main issues that prevent Sak from clinical applications. Herein, we explore two modifications of Sak to improve its therapeutic potential. Covalently attaching PEG (polyethylene glycol) polymers to therapeutic proteins has been widely applied to improve their pharmacokinetic properties, resulting in several FDA-approved protein-based drugs. Although PEGylation of Sak has previously been investigated, the effects of PEGylation on the 3D structure of Sak have not been studied in detail. In the first part of this research project, site-specific PEGylation of the truncated version of Sak was performed in the immunogenic region of the protein, and three covalent PEGylated derivatives of the protein were prepared. NMR studies showed a slight structural perturbation upon PEGylation, mostly close to the PEGylation site, and a direct relationship between the hydrodynamic radius of the PEGylated protein and the PEG size. Together, the results suggest that PEG and Sak∆10 move relatively independently from each other. As a second approach, we introduce cyclic Sak (cyc-Sak), a novel form of staphylokinase with higher stability and improved plasminogen activation activity. Using an Ssp GyrB split intein, the N- and C-terminal ends of the linear Sak were connected by a peptide bond, rendering the protein into a cyclic form (cyc-Sak). This structural modification was generated at the protein expression level in Escherichia coli, and the cyclic protein could be purified by common chromatography techniques. Successful backbone cyclization was confirmed by NMR spectroscopy of the 13C,15N-labeled cyc-Sak and by chemical cleavage assays. Our studies show that mono-PEGylation and intein-mediated backbone cyclization of Sak are powerful strategies to improve its therapeutic potential. These approaches could be combined to facilitate the design of protein-based medications when the stability of therapeutic proteins is an obstacle to their clinical application.
dc.identifier.citation	Baharian, A. (2023). Improving the therapeutic potential of staphylokinase, a potent thrombolytic agent (Master's thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca.
dc.identifier.uri	https://hdl.handle.net/1880/116582
dc.identifier.uri	https://dx.doi.org/10.11575/PRISM/41425
dc.language.iso	en
dc.publisher.faculty	Graduate Studies
dc.publisher.institution	University of Calgary
dc.rights	University of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission.
dc.subject	Staphylokinase
dc.subject	protein cyclization
dc.subject	protein PEGylation
dc.subject	intein
dc.subject	NMR Spectroscopy
dc.subject.classification	Biochemistry
dc.title	Improving the therapeutic potential of staphylokinase, a potent thrombolytic agent
dc.type	master thesis
thesis.degree.discipline	Biological Sciences
thesis.degree.grantor	University of Calgary
thesis.degree.name	Master of Science (MSc)
ucalgary.thesis.accesssetbystudent	I require a thesis withhold – I need to delay the release of my thesis due to a patent application, and other reasons outlined in the link above. I have/will need to submit a thesis withhold application.