Submitted to: Protein Expression and Purification
Publication Type: Peer reviewed journal
Publication Acceptance Date: 11/25/2002
Publication Date: 4/1/2003
Citation: SZURMAK, B., STROKOVSKAYA, L., MOONEY, B.P., RANDALL, D.D., MIERNYK, J.A. EXPRESSION AND ASSEMBLY OF ARABIDOPSIS THALIANA PYRUVATE DEHYDROGENASE IN INSECT CELL CYTOPLASM. PROTEIN EXPRESSION AND PURIFICATION. 2003. v. 28. p. 357-361. Interpretive Summary: Respiration is the use of energy by living cells to do work. Both growth and reproduction are directly coupled to rates of respiration. As a result, respiration must be carefully controlled or wasted energy would decrease crop yields and reduce agricultural productivity. The control of respiration in plant cells is a subject of ongoing study. A protein that is critical in the regulation of respiration was isolated from mouse eared cress plants, and studied. The protein comprises two copies each of two different components which must be correctly assembled in order to be biologically active. A method was developed to produce a completely assembled and biologically active form of the protein in insect cells. This information will be important to researchers in their attempts to increase agricultural productivity by improving the control of plant cell respiration, and to other plant scientists who will try to design more efficient crop plants through either classical breeding or biotechnology.
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-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 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 alpha2beta2 heterotetramer. Recombinant E1 was able to decarboxylate 1-14C-pyruvate, and was a substrate for in vitro phosphorylation by E1-kinase.