Production of an enzymatically active E1 component of human pyruvate dehydrogenase complex in Escherichia coli: supporting role of E1 beta subunit in E1 activity.
A co-expression plasmid containing the coding sequence of both the human liver pyruvate dehydrogenase (PDH) E1 alpha and E1 beta subunits was constructed. Functionally active PDH E1 protein was produced when this co-expression plasmid was introduced into the host Escherichia coli cell, BL21 (DE3)/plysS. In contrast, the production of E1 alpha alone resulted in a catalytically inactive protein, suggesting an important role of the E1 beta subunit in constituting enzyme activity. The PDH E1 protein produced in E. coli was capable of being phosphorylated by PDH-specific kinase. This co-expression system will provide a useful tool for studying the biochemical properties of human PDH E1.
Jeng
J
J
Laboratory of Neurogenetics, National Institute on Alcohol Abuse and Alcoholism, Rockville, MD.
Huh
T L
TL
Song
B J
BJ
D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D017931:DNA Primers; D004926:Escherichia coli; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods; D011768:Pyruvate Dehydrogenase Complex / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping
D017931:DNA Primers; D046911:Macromolecular Substances; D011768:Pyruvate Dehydrogenase Complex; D011994:Recombinant Proteins