A point mutation that decreases the thermal stability of human interferon {gamma} |
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Authors: | Lunn, C.A. Fossetta, J. Murgolo, N. Zavodny, P. J. Lundell, D. Narula, S.K. |
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Affiliation: | 1Departments of Biotechnology/Molecular Biology Bloomfield, NJ 07003, USA 2Departments of Biochemistry, Schering-Plough Research Institute Bloomfield, NJ 07003, USA |
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Abstract: | We have identified a mutation of human gamma-interferon (IFN)causing a temperature-sensitive phenotype. We used a randomizedoligonucleotide to mutagenize a synthetic human IFN gene, thenscreened the resulting mutants produced in Escherichia colifor proteins with altered biological activity. One mutant proteinselected for detailed characterization exhibited < 0.3% ofthe specific biological activity of native IFN in an antiviralactivity assay performed at 37°C. However, the protein boundthe human IFN receptor with native efficiency at 4°C. Sequencingthe plasmid DNA encoding this protein snowed that the mutationchanged the lysine residue at amino acid 43 to glutamic acid(IFN/K43E). Site-specific mutagenesis at amino acid 43 showedthat this protein's phenotype resulted from positioning a negativecharge at position 43. Structural characterization of IFN/K43Eusing CD demonstrated that the protein had native conformationat 25°C, but assumed an altered conformation at 37°C.IFN/K43E in this altered conformation bound poorly to the IFNreceptor at 37°C, providing a rationale for the mutant'sdecreased antiviral activity. |
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Keywords: | interferon /math/gamma.gif" ALT=" {gamma}" BORDER=" 0" >/ oligonucleotide-directed mutagenesis/ protein conformation/ receptor binding/ temperature-sensitive mutants |
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