cDNA cloning, expression in Escherichia coli and purification of human 6-pyruvoyl-tetrahydropterin synthase.
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.
Ashida
A
A
Department of Medical Chemistry, Osaka Medical College, Japan.
Hatakeyama
K
K
Kagamiyama
H
H
D018528:ATP-Binding Cassette Transporters; D000429:Alcohol Oxidoreductases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D015951:Factor Xa / Q000378:metabolism; D006801:Humans; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D019761:Phosphorus-Oxygen Lyases; D012333:RNA, Messenger / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D017422:Sequence Analysis, DNA
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli; D000429:Alcohol Oxidoreductases; D015951:Factor Xa; D019761:Phosphorus-Oxygen Lyases; C043497:6-pyruvoyltetrahydropterin synthase