Expression and purification of pyruvate transporting human mitochondrial pyruvate carrier (MPC) protein complex for structural studies

Abstract: Pyruvate is one of the main molecules needed for the citric acid cycle, which produces energy in the form of ATP. The citric acid cycle reaction occurs in the mitochondrial matrix, and for pyruvate to participate in the reaction, it needs to be transported from the cytosol across the mitochondrial membranes into the mitochondrial matrix. A special membrane protein called the mitochondrial pyruvate carrier (MPC) transports pyruvate across the inner membrane of the mitochondria, while pores in the outer mitochondrial membrane allows smaller molecules to diffuse over it. The MPC is made of two proteins, MPC1, and MPC2. The aim of this project is to characterize the structure and functionality of the mitochondrial pyruvate carrier. In this study, the Hu-MPC1 and Hu-MPC2 proteins were expressed separately in Pichia pastoris, and then individual and combined protein assays were performed. The assays include SDS-PAGE, Native-PAGE, Western Blot, and Mass spectrometry. To enable chromatography and assays of the proteins, the Hu-MPC1 was tagged with a Strep-tag II at the C-terminal and the Hu-MPC2 was tagged with a His-tag at the C-terminal. The results suggests that a heterodimer complex was formed between Hu-MPC1 and Hu-MPC2. Further research is desired with Hu-MPC1 induction for 24 hours to see if it is possible to produce a larger amount of protein. Further assays are necessary with Hu-MPC1 and Hu-MPC2 and as a complex, to gain more knowledge regarding the complex formation of the two subunits and their dimerization capabilities. Lastly, further studies are essential with purification optimization of Hu-MPC1 and Hu-MPC2, and usage of Cryo-EM or X-ray crystallography to solve the structure of the Hu-MPC complex and subunits.