Expression, purification and structural analysis of functional GABA transporter 1 using the baculovirus expression system
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Published on: August 20, 2018
Jing Hu1,2, Chris Weise3, Christoph Böttcher4
1Wuxi School of Medicine, Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Lihu Avenue 1800, 214122, Wuxi, China.
Abstract:
The γ-aminobutyric acid (GABA) transporter 1 (GAT1) belongs to a family of Na+ and Cl--coupled transport proteins and possesses 12 putative transmembrane domains. To perform structural analyses of the GAT1 protein, the GAT1/green fluorescent protein (GFP) fusion protein was functionally expressed in insect Sf9 cells by the BAC-TO-BAC® baculovirus expression system. A two-step procedure to purify the GAT1/GFP fusion protein from insect Sf9 cells has been established and involves immunoaffinity chromatography using self-prepared anti-GFP antibodies and size-exclusion fast protein liquid chromatography (SE-FPLC). A yield of 200-300 μg of the GAT1/GFP protein could be purified from 400-600 mL of infected Sf9 cells. The purified protein was analyzed by transmission electron microscopy (TEM), which revealed that the GAT1/GFP fusion protein was isolated in its monomeric form.
