Functional expression of milligram quantities of the synthetic human serotonin transporter gene in a tetracycline-inducible HEK293 cell line

• Published in: Protein expression and purification 2011-04, Vol.76 (2), p.211-220
• Main Authors: Takayama, Hidehito, Sugio, Shigetoshi
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Blotting, Western; Chromatography, Affinity; Cloning, Molecular; Digitonin; Electrophoresis, Polyacrylamide Gel; Glycosylation; HEK293 Cells; Humans; Imipramine - analysis; Imipramine - metabolism; Intracellular Space - metabolism; Intrinsically disordered region; Microscopy, Fluorescence; Molecular Sequence Annotation; Molecular Sequence Data; Oligopeptides; Peptides - genetics; Peptides - metabolism; Protein Conformation; Protein Engineering - methods; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Serotonin Plasma Membrane Transport Proteins - biosynthesis; Serotonin Plasma Membrane Transport Proteins - chemistry; Serotonin Plasma Membrane Transport Proteins - genetics; Serotonin Plasma Membrane Transport Proteins - metabolism; Serotonin transporter; Synthetic gene; Tandem Mass Spectrometry; Tetracycline - pharmacology; Tetracycline-inducible HEK293 cells; Tritium - analysis
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2010.11.015

The serotonin transporter (SERT), a member of the solute carrier 6 family, is responsible for reuptake of the monoamine neurotransmitter serotonin (5-hydroxytryptamine) from the synaptic cleft on the neural cells, and a vital target for several antidepressants. To investigate biophysical studies of this pharmacologically relevant transporter, we developed a mammalian expression system with tetracycline-inducible HEK293 cells using synthetic human SERT genes produced by PCR-based self-assembly method. Codon-optimization of this de novo constructed genes and construction of stable cell lines improved expression 3.5-fold and single-step immunoaffinity purification with FLAG-epitope tag yielded around one milligram functional SERT per liter culture medium assessed by [3H] imipramine ligand binding. Some characterizations including electrospray ionization MS/MS analysis, subcellular localization and cellular-uptake assay demonstrated that expressed human SERT was properly expressed, folded and fully functional. The long cytosolic N-terminal of SERT was predicted as containing ‘intrinsically disordered region (IDR)’ (∼85 residues) by DISOPRED2 program. We engineered this salient region by step-wise truncation and ligand binding assay determined that dissociation constant for a series of de novo designed truncation constructs was close to the one for full-length wild type SERT. Our expression platform using synthetic codon-optimized gene and mammalian stable cell lines is feasible to produce milligram-scale functional membrane transporter for further biophysical and biochemical studies.