Abstract
A self-cleaving element for use in bioseparations has been derived from a naturally occurring, 43 kDa protein splicing element (intein) through a combination of protein engineering and random mutagenesis. A mini-intein (18 kDa) previously engineered for reduced size had compromised activity and was therefore subjected to random mutagenesis and genetic selection. In one selection a mini-intein was isolated with restored splicing activity, while in another, a mutant was isolated with enhanced, pH-sensitive C-terminal cleavage activity. The enhanced-cleavage mutant has utility in affinity fusion-based protein purification. These mutants also provide new insights into the structural and functional roles of some conserved residues in protein splicing.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Amino Acid Sequence
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Chromatography, Affinity
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DNA Gyrase
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DNA Topoisomerases, Type II / genetics
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DNA Topoisomerases, Type II / isolation & purification
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Endopeptidases / genetics
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Endopeptidases / metabolism
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Leucine / genetics
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Models, Molecular
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Molecular Sequence Data
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Mutagenesis*
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Mycobacterium / genetics
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Protein Engineering / methods*
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Protein Precursors / metabolism*
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Protein Splicing / genetics*
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Rec A Recombinases / genetics
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Recombinant Proteins / isolation & purification*
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Thymidylate Synthase / genetics
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Thymidylate Synthase / isolation & purification
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Valine / genetics
Substances
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Protein Precursors
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Recombinant Proteins
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Thymidylate Synthase
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Rec A Recombinases
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Endopeptidases
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DNA Gyrase
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DNA Topoisomerases, Type II
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Leucine
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Valine