Development of an Expression and Purification System of Recombinant Fibrinogen Towards Studying the Impact of N-linked Glycans on Fibrinogen Structure and Polymerization

ECU Author/Contributor (non-ECU co-authors, if there are any, appear on document)

Grega Popovic (Creator)

Institution

East Carolina University (ECU )

Web Site: http://www.ecu.edu/lib/

Abstract: Fibrinogen is a 340 kDa glycoprotein that is capable of creating an insoluble clot during a process called fibrin polymerization. This is initiated with the conversion of fibrinogen to fibrin monomer conversion through cleavage and release of fibrinopeptides A and B, by an enzyme called thrombin. As proof-of-principle, functional studies on the WT protein from commercial fibrinogen were conducted in which the N-linked glycans were removed by PNGase F or processed with neuraminidase. Turbidity and fibrinopeptide release assays in conjunction were employed to characterize the effects of N-linked glycans on fibrin polymerization. In order to fully depict the fibrinogen structure and function of its glycans, a functional expression and purification system is necessary. A recombinant system using transient transfection methods in HEK and CHO cells is being developed with varied conditions such as different PEI:DNA ratios, use of circular versus linear DNA, and performing the transfection with suspended vs adherent cells. A novel synthetic peptide Fmoc-GPRPFPAWK, bound to NHS-activated Sepharose 4 Fast Flow resin is introduced as a viable affinity based purification technique. This will enable future studies linked to site-specific glycans.

Additional Information

Publication

Thesis

Language: English

Date: 2023

Subjects

fibrin;transient expression;purification;hek;cho;n-linked glycans;fibrin polymerization

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