Asp295 Stabilizes the Active-Site Loop Structure of Pyruvate Dehydrogenase, Facilitating Phosphorylation of Ser292 by Pyruvate Dehydrogenase-Kinase
Figure 2
Expression and purification of A. thaliana pyruvate dehydrogenase. (a) Bacterial expression of AtPDC E1α and E1β. An SDS gel of total proteins from uninduced E. coli BL21 (DE3) cells containing pT7-7:AtE1α; insoluble (I.F.) and soluble (S.F.) fractions from induced cells; total proteins from uninduced cells containing pET28:AtE1β; insoluble and soluble fractions from induced cells. The positions of AtPDC E1α and E1β are indicated by arrows. (b) An SDS gel of a typical 2-plasmid AtPDC E1 α plus β coexpression and purification profile using the HiTrap chelating column charged with 0.1 M NiSO4; insoluble fraction, soluble fraction, flow through (F.T.). The position of AtPDC E1β in the soluble fraction is indicated by an arrow. (c) A Coomassie-stained SDS gel of AtPDC E1 after immobilized metal affinity chromatography. (d) Immunoblot analysis of AtPDC E1. The E1α subunit was detected with monoclonal anti-E1α antibodies, and the E1β subunit was detected with monoclonal anti-FLAG antibodies. The positions of molecular mass markers are indicated on the left for (a) and (b). All gels (except the Immunoblot) were stained with Coomassie Blue.
