Abstract
cDNA clones for human 6-pyruvoyl-tetrahydropterin synthase, the second enzyme in the biosynthetic pathway of tetrahydrobiopterin, were isolated from a human Molt-4 cell cDNA library by cross-hybridization with a rat cDNA. One cDNA clone contained the entire coding sequence of 435 base pairs. The cDNA was expressed in Escherichia coli using the expression vector pMAL as a fusion protein with maltose-binding protein. After affinity purification through its maltose-binding protein domain, the fusion protein was digested by factor Xa at a specific cleavage site inserted between the domains. The main product was a protein species with a native molecular mass of 90 kDa and a subunit molecular mass of 17 kDa, and the molecular masses and its kinetic properties were similar to those of the human enzyme purified from the liver.
MeSH terms
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ATP-Binding Cassette Transporters*
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Alcohol Oxidoreductases / biosynthesis
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Alcohol Oxidoreductases / genetics*
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Alcohol Oxidoreductases / isolation & purification*
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Amino Acid Sequence
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Base Sequence
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Carrier Proteins / biosynthesis
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Carrier Proteins / genetics
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Cloning, Molecular
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Escherichia coli / genetics
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Escherichia coli Proteins*
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Factor Xa / metabolism
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Humans
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Maltose-Binding Proteins
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Molecular Sequence Data
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Monosaccharide Transport Proteins*
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Phosphorus-Oxygen Lyases*
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RNA, Messenger / genetics
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Recombinant Fusion Proteins / biosynthesis
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Sequence Analysis, DNA
Substances
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ATP-Binding Cassette Transporters
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Carrier Proteins
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Escherichia coli Proteins
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Maltose-Binding Proteins
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Monosaccharide Transport Proteins
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RNA, Messenger
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Recombinant Fusion Proteins
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maltose transport system, E coli
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Alcohol Oxidoreductases
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Factor Xa
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Phosphorus-Oxygen Lyases
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6-pyruvoyltetrahydropterin synthase
Associated data
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GENBANK/D17400
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GENBANK/D37827
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GENBANK/L16877
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GENBANK/L16878
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GENBANK/L19108
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GENBANK/L19109
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GENBANK/L19111
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GENBANK/L19112
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GENBANK/L19956
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GENBANK/L19957