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Expression, purification and structural analysis of functional GABA transporter 1 using the baculovirus expression system.

ABSTRACT: The ?-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 ?g 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.

SUBMITTER: Hu J

PROVIDER: S-EPMC5433199 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Expression, purification and structural analysis of functional GABA transporter 1 using the baculovirus expression system.

Hu Jing J Weise Chris C Böttcher Christoph C Fan Hua H Yin Jian J

Beilstein journal of organic chemistry 20170511

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 ...[more]

PMID: 28546845

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