Abstract
We describe an activity-independent method for the selection of thermostable mutants of any protein. It is based on a fusion construct comprising the protein of interest and a thermostable antibiotic resistance reporter, in such a way that thermostable mutants provide increased resistance in a thermophile. We isolated thermostable mutants of three human interferons and of two enzymes to demonstrate the applicability of the system.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Substitution / physiology
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Escherichia coli / genetics
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Formate Dehydrogenases / chemistry
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Formate Dehydrogenases / genetics
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Hot Temperature*
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Humans
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Interferon-alpha / chemistry
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Interferon-alpha / genetics
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Interferon-beta / chemistry
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Interferon-beta / genetics
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Interferon-gamma / chemistry
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Interferon-gamma / genetics
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Kanamycin / pharmacology
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Lipase / chemistry
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Lipase / genetics
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Nucleotidyltransferases / chemistry
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Nucleotidyltransferases / genetics
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Nucleotidyltransferases / metabolism
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Peptide Fragments / chemistry
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Peptide Fragments / genetics
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Protein Engineering / methods*
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Protein Folding
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Proteins / chemistry*
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Proteins / genetics
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Proteins / metabolism
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Recombinant Proteins / chemistry
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Recombinant Proteins / metabolism
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Thermus thermophilus / drug effects
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Thermus thermophilus / genetics
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Thermus thermophilus / growth & development
Substances
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Interferon-alpha
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Peptide Fragments
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Proteins
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Recombinant Proteins
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Kanamycin
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Interferon-beta
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Interferon-gamma
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Formate Dehydrogenases
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Nucleotidyltransferases
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kanamycin nucleotidyltransferase
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Lipase