Comparison of seven different heterologous protein expression systems for the production of the serotonin transporter

Abstract

The rat serotonin transporter (rSERT) is an N-glycosylated integral membrane protein with 12 transmembrane regions; the N-glycans improve the ability of the SERT polypeptide chain to fold into a functional transporter, but they are not required for the transmembrane transport of serotonin per se. In order to define the best system for the expression, purification and structural analysis of serotonin transporter (SERT), we expressed SERT in Escherichia coli, Pichia pastoris, the baculovirus expression system and in four different stable mammalian cell lines. Two stable cell lines that constitutively expressed SERT (Imi270 and Coca270) were constructed using episomal plasmids in HEK293 cells expressing the EBNA-1 antigen. SERT expression in the three different inducible stable mammalian cell lines was induced either by a decrease in temperature (cell line pCytTS-SERT), the addition of tetracycline to the growth medium (cell line T-REx-SERT) or by adding DMSO which caused the cells to differentiate (cell line MEL-SERT). All the mammalian cell lines expressed functional SERT, but SERT expressed in E. coli or P. pastoris was nonfunctional as assessed by 5-hydroxytryptamine uptake and inhibitor binding assays. Expression of functional SERT in the mammalian cell lines was assessed by an inhibitor binding assay; the cell lines pCytTS-SERT, Imi270 and Coca270 contained levels of functional SERT similar to that of the standard baculovirus expression system (250,000 copies per cell). The expression of SERT in induced T-REx-SERT cells was 400,000 copies per cell, but in MEL-SERT it was only 80,000 copies per cell. All the mammalian stable cell lines expressed SERT at the plasma membrane as assessed by [3H]-5-hydroxytryptamine uptake into whole cells, but the V(max) for the T-Rex-SERT cell line was 10-fold higher than any of the other cell lines. It was noticeable that the cell lines that constitutively expressed SERT grew extremely poorly, compared to the inducible cell lines whose growth rates were similar to the parental cell lines when not induced. In addition, the cell lines MEL-SERT, Imi270 and T-REx-SERT all expressed fully N-glycosylated SERT and no unglycosylated inactive protein, in contrast to the baculovirus expression system where the vast majority of expressed SERT was unglycosylated and nonfunctional.

Heterologous protein production using the Pichia pastoris expression system

“…Additionally, it had been produced in reasonable quantities (3 mg/g cell mass), enough for the purpose of structural studies. Other attempts at producing mammalian membrane proteins [109] in P. pastoris have not had the same success. The rat serotonin transporter, rSERT, was found to be misfolded and glycosylated when produced in a protease-deficient P. pastoris strain, as well as having a very low binding activity.…”

Comparative potencies of 3,4‐methylenedioxymethamphetamine (MDMA) analogues as inhibitors of [3H]noradrenaline and [3H]5‐HT transport in mammalian cell lines

“…The original cell line was created and supplied by Dr Chris Tate (University of Cambridge, UK). Full details of the transfection procedure may be found in Tate et al (2003). Briefly, rat SERT cDNA was used as a template to generate a PCR fragment containing the complete coding sequence but lacking the stop codon.…”

“…The complete insert of the plasmid was checked by DNA sequencing. The plasmid was used to transfect an HEK293 cell line (T-REx-293) that expressed the tetracycline repressor protein, Tet, as described (Tate et al, 2003). Expression of SERT was confirmed by western blotting, inhibitor-binding assays on crude membrane preparations, [ 3 H]5-HT uptake assays into whole cells and by confocal microscopy of immunostained cells.…”

“…Consequently, it was decided to perform this investigation using NET and SERT. The rat phaeochromocytoma (PC12) cell line was chosen as the experimental model for the noradrenergic system (Greene and Tischler, 1976), whereas the T-REx SERT HEK 293 cell line was selected as the experimental model for the 5-hydroxytryptaminergic system (Tate et al, 2003).…”

LeuT: A prokaryotic stepping stone on the way to a eukaryotic neurotransmitter transporter structure

“…37 Despite these momentous strides, the need for a structural paradigm for the interpretation of this wealth of functional data remained. Valiant attempts have been made to express, 38 solubilize 39 and purify representative SLC6 proteins to near homogeneity, [40][41][42] but these proteins have proven labile and, thus, have yet to unveil their atomic secrets. Proteins from prokaryotic organisms are generally more stable than their eukaryotic counterparts and can be expressed at much higher levels.…”