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Modifying Baculovirus Expression Vectors to Produce Secreted Plant Proteins in Insect Cells
Published on: August 20, 2018
Expression and assembly of Arabidopsis thaliana pyruvate dehydrogenase in insect cell cytoplasm
Blanka Szurmak1, Ludmila Strokovskaya, Brian P Mooney
1Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5A, 02_106 Warsaw, Poland.
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-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.
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