Mans (2002) hypothesized that the two domains of savignin interact with each other, giving a globular form in the absence of thrombin. Binding of the C-terminal domain of the inhibitor to the fibrinogen-binding site of thrombin leads to the dissociation of the domains. This would yield an extended conformation that would allow binding of the N-terminal residues of the N-terminal domain to thrombin’s active site. To test this hypothesis, both theoretical and experimental approaches were employed to determine the molecular dimensions of uncomplexed savignin. In the theoretical approach, the hydrodynamic radius (Rh) of the extended form of savignin was calculated from the crystal structure data of the thrombin-ornithodorin complex, and found to be 2.319 nm. With the same programme, based on the crystal structure data for bikunin, a protein in which both domains are closely associated, the Rh value for the compact form of savignin was estimated as 1.96 nm. Using the equation that relates Rh to molecular mass, a value of 1.84 nm was calculated for savignin (12 430 Da). In the experimental approach, the SEC of salivary gland extracts, using lysozyme (Rh = 1.99 nm) as standards and chrymotrypsinogen (Rh = 2.31nm) indicated that uncomplexed savignin exists in both the globular and extended conformations. However, the majority of inhibitory activity was associcated with the extended form. Heat stability assays as well as SDS-PAGE experiments indicated the possible existence of the compact form of savignin. Generation of adequate amounts of savignin will allow further structural studies, to determine the structure of savignin in the uncomplexed form and in complex with thrombin. Expression of full length savignin and the separate N- and C-domains will facilitate further kinetic analysis. In the recombinant production of savignin, various factors were investigated: cell-lines, transformation efficiency, induction times, purification strategy and protease cleavage of expressed fusion protein. Even though large quantities of expressed fusion protein were obtained, cleavage of the target protein and its separation from the fusion partner by enzymatic means presented a major hurdle. Expression of Nsav was not observed and is most likely as a result of misfolding of the recombinant form. Due to the probable non-specific cleavage of the fusion protein, switching the prokaryotic expression system to other expression systems, like yeast- or baculovirus-insect cell-expression systems, is warranted.
Dissertation (MSc (Biochemistry))--University of Pretoria, 2007.