Abstract
The bi-functional enzyme, adenosylcobinamide kinase/adenosylcobinamide-phosphate guanylyltransferase (CobU), is involved in the biosynthesis of cobalamin in Salmonella typhimurium, and, therefore, can be used for the in vitro synthesis of analogs of B(12). Previously, five different steps were required to purify the recombinant enzyme from Escherichia coli. Here, we describe the cloning, sequencing, and expression of the cobU gene from S. typhimurium ATCC 19585 and, without introducing a purification tag sequence to the N- or C-terminus of the recombinant enzyme, a new single-step purification method based on hydrophobic interaction chromatography.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Chromatography, Agarose / methods
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Cloning, Molecular
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Escherichia coli / genetics
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Gene Expression / genetics
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Genetic Vectors / genetics
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Hydrophobic and Hydrophilic Interactions
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Multienzyme Complexes / genetics
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Multienzyme Complexes / isolation & purification
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Multienzyme Complexes / metabolism*
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Nucleotidyltransferases / genetics
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Nucleotidyltransferases / isolation & purification
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Nucleotidyltransferases / metabolism*
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Pentosyltransferases / genetics
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Pentosyltransferases / isolation & purification
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Pentosyltransferases / metabolism*
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Recombinant Proteins / biosynthesis*
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Recombinant Proteins / chemistry
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Recombinant Proteins / isolation & purification
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Salmonella typhimurium / enzymology*
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Salmonella typhimurium / genetics
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Sepharose / analogs & derivatives
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Sepharose / chemistry
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Sequence Analysis, DNA
Substances
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Multienzyme Complexes
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Recombinant Proteins
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phenyl-sepharose
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Sepharose
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Pentosyltransferases
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nicotinate-nucleotide-dimethylbenzimidazole phosphoribosyltransferase
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Nucleotidyltransferases