Functional expression, purification and high sequence coverage mass spectrometric characterization of human excitatory amino acid transporter EAAT2.

ABSTRACT: The glial excitatory amino acid transporter 2 (EAAT2) mediates a majority of glutamate re-uptake in human CNS and, consequently, is associated with a variety of signaling and pathological processes. While our understanding of the function, mechanism and structure of this integral membrane protein is increasing, little if any mass spectrometric (MS) data is available for any of the EAATs specifically, and for only a few mammalian plasma membrane transporters in general. A protocol to express and purify functional EAAT2 in sufficient quantities to carry out MS-based peptide mapping as needed to study ligand-transporter interactions is described. A 6xHIS epitope was incorporated into the N-terminus of human EAAT2. The recombinant protein was expressed in high levels in mammalian HEK 293T cell

SUBMITTER: Ye R 

PROVIDER: S-EPMC4356123 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature