Submitted to: Protein Expression and Purification
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/5/2000
Publication Date: 7/1/2000
Citation: MORENO, J.I., DAVID, N.R., MIERNYK, J.A., RANDALL, D.D. PISUM SATIVUM MITOCHONDRIAL PYRUVATE DEHYDROGENASE CAN BE ASSEMBLED AS A FUNCTIONAL ALPHA2BETA2 HETEROTETRAMER IN THE CYTOPLASM OF PICHIA PASTORIS. PROTEIN EXPRESSION AND PURIFICATION. 2000. V. 19(2). P. 276-273. Interpretive Summary: Respiration is the use of energy by living cells to do work. Both growth and reproduction are affected by 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 important in the regulation of respiration was isolated from garden peas, and studied. Comparisons were made with a similar protein from animals and microbes in order to predict characteristics that might be important in control of respiration. A method was developed to produce a biologically active form of the protein in yeast cells. This information will be important to researchers in their attempts to increase agricultural productivity by altering the control of plant cell respiration, and to other plant scientists who will try to design more efficient crop plants through either rclassical breeding or biotechnology.
Technical Abstract: Pea (Pisum sativum) mitochondrial pyruvate dehydrogenase (E1) was produced by co-expression of the mature alpha and beta subunits in the cytoplasm of the yeast Pichia pastoris. Size exclusion chromatography of recombinant E1, using a Superose 12 column, yielded a peak at Mr 160,000 that contained both alpha and beta subunits as well as E1 activity. This corresponds to the size of native alpha2beta2 E1. Recombinant Eq alpha(His6)-E1beta was purified by affinity chromatography using immobilized Ni+, with a yield of 2.8 mg/L. The pyruvate decarboxylating activity of recombinant E1 was dependent upon added Mg2+ and thiamin-pyrophosphate, and was enhanced by the oxidant potassium ferricyanide. Native pea mitochondrial E1-kinase catalyzed phosphorylation of Ser residues in the alpha-subunit of recombinant E1, with concomitant loss of enzymatic activity. Thus, mitochondrial pyruvate dehydrogenase can be assembled in the cytoplasm of P. pastoris into an alpha2beta2 hetrotetramer that is both catalytically active and competent for regulatory phosphorylation.