Expression and assembly of Arabidopsis thaliana pyruvate dehydrogenase in insect cell cytoplasm.
A vector was constructed for expression of Arabidopsis thaliana mitochondrial pyruvate dehydrogenase (E1) in the cytoplasm of Trichoplusia ni cells. The construct pDDR101 comprises the mature-E1alpha coding sequence under control of the Polh promoter, plus the mature-E1beta coding sequence under control of the p10 promoter. The E1alpha sequence was engineered to include an N-terminal His-tag. When protein samples were subjected to immobilized metal ion affinity chromatography, the alpha- and beta-subunits co-eluted, indicating association. When the recombinant protein sample was analyzed further by gel permeation chromatography, it was demonstrated that a significant amount eluted at a size consistent with assembly into an alpha2beta2 heterotetramer. Recombinant E1 was able to decarboxylate [1-14C]pyruvate and was a substrate for in vitro phosphorylation by E1-kinase.
Szurmak
Blanka
B
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5A, 02_106 Warsaw, Poland.
Strokovskaya
Ludmila
L
Mooney
Brian P
BP
Randall
Douglas D
DD
Miernyk
Jan A
JA
D000818:Animals; D017360:Arabidopsis / Q000201:enzymology*; D002384:Catalysis; D002460:Cell Line; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D007313:Insecta / Q000166:cytology / Q000235:genetics*; D008928:Mitochondria / Q000201:enzymology; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D011768:Pyruvate Dehydrogenase Complex / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera
D011768:Pyruvate Dehydrogenase Complex; D011994:Recombinant Proteins; D011494:Protein Kinases