LEADER 03655cam 2200625Ii 4500001 on1257305633 003 OCoLC 005 20220211123111.0 006 m o d 007 cr unu|||||||| 008 210622s2021 ncuab obm 000 0 eng d 035 (Sirsi) o1257305633 035 (OCoLC)1257305633 040 ERE |beng |erda |cERE |dOCLCO |dOCLCQ |dOCLCF |dOCLCO |dERE |dUtOrBLW 049 EREE 090 QP93.5 100 1 Popovic, Grega, |eauthor. |?UNAUTHORIZED 245 10 Development of an expression and purification system of recombinant fibrinogen towards studying the impact of N-linked glycans on fibrinogen structure and polymerization / |cby Grega Popovic. 264 1 [Greenville, N.C.] : |b[East Carolina University], |c2021. 300 1 online resource (109 pages) : |billustrations (chiefly color), maps 336 text |btxt |2rdacontent 337 computer |bc |2rdamedia 338 online resource |bcr |2rdacarrier 347 text file 347 |bPDF 347 |c3.345 MB 538 System requirements: Adobe Reader. 538 Mode of access: World Wide Web. 502 |bM.S. |cEast Carolina University |d2021 500 Presented to the faculty of the Department of Chemistry. 500 Advisor: Adam R. Offenbacher 500 Title from PDF t.p. (viewed February 9, 2022). 520 3 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. 504 Includes bibliographical references. 650 0 Fibrinogen. |=^A835115 650 0 Recombinant proteins. |=^A719573 650 0 Proteins |xConformation. |=^A312178 653 fibrin 653 transient expression 653 purification 653 hek 653 cho 653 n-linked glycans 653 fibrin polymerization 655 7 Academic theses. |2fast |0(OCoLC)fst01726453 655 7 Academic theses. |2lcgft 700 1 Offenbacher, Adam R., |edegree supervisor. |?UNAUTHORIZED 710 2 East Carolina University. |bDepartment of Chemistry. |?UNAUTHORIZED 856 40 |uhttp://hdl.handle.net/10342/9134 |zAccess via ScholarShip 949 |owjh 994 C0 |bERE 596 1 4 998 5636999