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1996
Journal Article
Titel
Engineered disulfide bonds in recombinant human interferon-gamma. The impact of the N-terminal helix A and the AB-loop on protein stability
Abstract
Insertion sites for cysteines with optimal stereochemistry for the formation of unstrained disulfide bridges were identified in recombinant human interferon-gamma (rhu-IFN-gamma) by computer modelling. We have engineered two different disulfide cross-linked mutants, containing a pair of symmetry-related disulfide bonds, which stabilize the N-termini of both monomers of the homodimeric protein. Mutations E7C and S69C allow the formation of an intramonomer disulfide bond between helices A and D. In contrast, the A17C and H111C mutations lead to a covalent cross-link between both monomers. THE AB-loop is linked to helix F. The fluorescence properties of native and disulfide cross-linked proteins were studied as a function of guanidine hydrochloride concentration. Melting temperatures (Tm) were calculated from the decrease in CD ellipticity at 220nm. The induction of the antiviral effect was measured using A549 fibroblast cells infected with encephaomyocarditis virus.
Language
English