Comparison of seven different heterologous protein expression systems for the production of the serotonin transporter.
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.
Tate
Christopher G
CG
MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK. cgt@mrc-lmb.cam.ac.uk
Haase
Jana
J
Baker
Cara
C
Boorsma
Marco
M
Magnani
Francesca
F
Vallis
Yvonne
Y
Williams
D Clive
DC
D000818:Animals; D015153:Blotting, Western; D002352:Carrier Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D002460:Cell Line / Q000378:metabolism; D003460:Crystallization; D004926:Escherichia coli / Q000378:metabolism; D005786:Gene Expression Regulation; D005821:Genetic Techniques; D006031:Glycosylation; D006801:Humans; D008562:Membrane Glycoproteins / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D026901:Membrane Transport Proteins; D018613:Microscopy, Confocal; D009154:Mutation; D009419:Nerve Tissue Proteins; D010843:Pichia / Q000378:metabolism; D050486:Serotonin Plasma Membrane Transport Proteins
D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; C493230:SLC6A4 protein, human; D050486:Serotonin Plasma Membrane Transport Proteins