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Expression, purification and structural analysis of functional GABA transporter 1 using the baculovirus expression
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.
Beilstein Journal of Organic Chemistry
|May 27, 2017
Summary
Researchers purified the γ-aminobutyric acid (GABA) transporter 1 (GAT1) fused with green fluorescent protein (GFP). This purified GAT1/GFP protein was analyzed using transmission electron microscopy, revealing its monomeric form.
Area of Science:
- Biochemistry
- Molecular Biology
- Neuroscience
Background:
- γ-aminobutyric acid (GABA) transporter 1 (GAT1) is a key neurotransmitter transporter.
- GAT1 is a member of the Na+ and Cl--coupled transport protein family.
- Understanding GAT1 structure is crucial for its functional analysis.
Purpose of the Study:
- To develop a method for purifying the GAT1 protein for structural studies.
- To express and purify a GAT1/green fluorescent protein (GFP) fusion protein.
- To analyze the oligomeric state of the purified GAT1/GFP fusion protein.
Main Methods:
- Functional expression of GAT1/GFP fusion protein in insect Sf9 cells using a baculovirus system.
- Two-step purification involving immunoaffinity chromatography and size-exclusion FPLC.
- Transmission electron microscopy (TEM) for structural analysis.
Main Results:
- Successfully purified GAT1/GFP fusion protein from insect cells.
- Achieved a yield of 200-300 μg of purified protein from 400-600 mL of infected cells.
- TEM analysis confirmed the purified GAT1/GFP protein exists in a monomeric form.
Conclusions:
- Established an effective purification protocol for GAT1/GFP.
- The purified GAT1/GFP protein is suitable for further structural and functional investigations.
- The monomeric state of GAT1/GFP provides a basis for future structural determination.
