ARS | Publication request: EXPRESSION AND ASSEMBLY OF ARABIDOPSIS THALIANA PYRUVATE DEHYDROGENASE IN INSECT CELL CYTOPLASM

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: December 2, 2002
Publication Date: N/A

Technical Abstract: 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-E1 alpha coding sequence under control of the polh promoter, plus the mature-E1 beta coding sequence under control of the p10 promoter. The E1 alpha sequence was engineered to include a 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 further analyzed by gel permeation chromatography, it was demonstrated that a significant amount eluted at a size consistent with assembly into an alpha-2-beta-2 heterotetramer. Recombinant E1 was able to decarboxylate 1-14C-pyruvate, and was a substrate for in vitro phosphorylation by E1-kinase.