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.
R&D Strategy Department, Corporate Strategy Division, Mitsubishi Chemical Corporation, Yokohama, Japan.
Hidehito
H
Takayama
Shigetoshi
S
Sugio
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D007099:Imipramine / Q000032:analysis / Q000378:metabolism; D042541:Intracellular Space / Q000378:metabolism; D008856:Microscopy, Fluorescence; D058977:Molecular Sequence Annotation; D008969:Molecular Sequence Data; D009842:Oligopeptides; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D050486:Serotonin Plasma Membrane Transport Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D013752:Tetracycline / Q000494:pharmacology*; D014316:Tritium / Q000032:analysis
D009842:Oligopeptides; D010455:Peptides; D011993:Recombinant Fusion Proteins; C493230:SLC6A4 protein, human; D050486:Serotonin Plasma Membrane Transport Proteins; D014316:Tritium; C085706:FLAG peptide; D013752:Tetracycline; D007099:Imipramine