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Engineered disulfide bonds in recombinant human interferon-gamma. The impact of the N-terminal helix A and the AB-loop on protein stability


Protein engineering 9 (1996), No.10, pp.905-912
ISSN: 0269-2139
Journal Article
Fraunhofer IGB ()
Fraunhofer ITA ( Fraunhofer ITA) ()
computer modelling; computer simulation; disulfide; disulfide bridge; genetic recombination; human; interferon; interferon gamma; molecular biology; mutagenesis; site-directed mutagenesis; site-specific mutagenesis

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.