Functional expression of milligram quantities of the synthetic human serotonin transporter gene in a tetracycline-inducible HEK293 cell line
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 [(3)H] 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.