Overexpression of membrane proteins in mammalian cells for structural studies

Figures & data

Table I. Structures of mammalian membrane proteins determined from protein overexpressed in mammalian cells.

Membrane protein	Source	Cells used for protein production	Resolution of structure*	Reference
Ammonia transporter RhCG	Human	HEK293S(TetR)-GnTI^-	2.1 Å	(Gruswitz et al. 2010)
Rhodopsin N2C/D282C/D113Q	Bovine	HEK293S(TetR)-GnTI^-	3.0 Å	(Standfuss et al. 2011)
Rhodopsin N2C/D282C/M257Y	Bovine	HEK293S(TetR)-GnTI^-	3.3 Å	(Deupi et al. 2012)
Rhodopsin N2C/D282C	Bovine	COS-7	3.4 Å	(Standfuss et al. 2007)
Connexin a1 (gap junction)	Human	BHK	7.5 Å	(Unger et al. 1999)

*Structures were determined by X-ray diffraction except for the structure of connexin, which was determined by electron cryo-microscopy of 2-dimensional crystals that formed in BHK cells upon overexpression. There are numerous examples of low resolution structures determined by electron microscopy of membrane proteins isolated after expression in mammalian cells, but only examples of structures at better than 8 Å resolution are shown. Details of the expression systems and amounts of protein expressed are shown in Tables II and III.

Table II. Expression of membrane proteins using transient expression systems in mammalian cells.

Membrane protein	Promoter	Cell line	Scale up	Expression levels*	Amount purified	Reference
Semliki Forest Virus expression
Neurokinin 1 receptor	SFV subgenomic	BHK-21	–	40 pmol/mg	–	(Lundstrom et al. 1994)
Histamine receptor H2	SFV subgenomic	COS-7	–	80 pmol/mg	–	(Hoffmann et al. 2001)
α2β adrenergic receptor	SFV subgenomic	CHO	–	175 pmol/mg mp; 8.8 million c/c	–	(Sen et al. 2003)
Adenosine receptor A2A	SFV subgenomic	BHK-21	–	285 pmol/mg; 1–10 mg/l	–	(Hassaine et al. 2006)
Glutamate transporter GLT1	SFV subgenomic	BHK-21	Stirrer flasks (8 l) 1 million cells/ml	3.5 million c/c; 61 pmol/mg mp	0.3 mg	(Raunser et al. 2005)
Bradykinin B2 receptor	SFV subgenomic	BHK-21	–	11 pmol/mg; 0.2 mg/l	–	(Shukla et al. 2006a)
Aquaporin AQP3	SFV subgenomic	various	1 l glass roller bottles	∼ 0.01 mg/l	–	(Eifler et al. 2007)
Aquaporin AQP6	SFV subgenomic	various	–	<0.001 mg/l	–	(Eifler et al. 2007)
HCN2 channel	SFV subgenomic	various	–	∼ 10 mg/l (+ misfolded)	–	(Eifler et al. 2007)
Purine receptor P2X2	SFV subgenomic	various	–	∼0.5 mg/l	–	(Eifler et al. 2007)
Purine receptor P2Y2	SFV subgenomic	various	–	∼ 10 mg/l	–	(Eifler et al. 2007)
Vasopressin receptor V2	SFV subgenomic	various	–	∼ 1 mg/l	–	(Eifler et al. 2007)
Angiotensin receptor AT1	SFV subgenomic	BHK	–	32 pmol/mg	–	(Shukla et al. 2006b)
Muscarinic M3 receptor	SFV subgenomic	COS-7	–	27 pmol/mg	–	(Romero-Fernandez et al. 2011)
Adenovirus virus expression
TRH receptor	CMV	HeLa	–	2 million c/c	–	(Falck-Pedersen et al. 1994)
Vaccinia virus expression
Neuropeptide Y receptor	Vaccinia 11K promoter	HeLa	–	1 mg/l	–	(Walker et al. 1993)
Transient transfection from plasmid DNA
β2 adrenergic receptor	Adenovirus major late promoter	COS-1	–	18 pmol/mg	–	(Chelikani et al. 2006)
Rhodopsin mutant		COS-7	50 × 15 cm plates	n.r.	∼ 0.6 mg	(Standfuss et al. 2007)

*Shown are some examples of membrane proteins expressed in mammalian cells at high-levels or where there are interesting comparisons between expression systems. Further examples can be found in (Grisshammer and Tate 1995, Sarramegna et al. 2003). Expression levels are shown in units as supplied in the primary publication: c/c, copies per cell; mg mp, milligram of membrane protein; n.r., not recorded.

Table III. Expression of membrane proteins in stable mammalian cell lines.

Membrane protein	Promoter	Cell line	Scale up	Expression levels*	Amount purified	Ref
Constitutive expression in stable mammalian cell lines
β2 adrenergic receptor	CMV	CHO	–	200 pmol/mg	1.3 nmol (∼60 ug)	(Lohse 1992)
Glucagon receptor	CMV	BHK	–	78 pmol/mg	–	(Jelinek et al. 1993)
Serotonin transporter	CMV	293EBNA	–	290,000 c/c	–	(Tate et al. 2003)
Chemokine receptor D6	CMV	L1.2	1 mg/5 × 10^9 cells	2.5 million c/c	–	(Blackburn et al. 2004)
Serotonin receptor 5HT2c	CMV	HEK293T	–	140 pmol/mg; 3 million c/c	–	(Mancia et al. 2004)
Bradykinin receptor B2	CMV	HEK293S	–	3 million c/c; 60 pmol/mg mp	–	(Camponova et al. 2007)
Chemokine receptor CCR1	CMV	Flp-In 293	–	Relative fluorescence	–	(Allen et al. 2009)
Inducible expression in stable mammalian cell lines
Serotonin transporter	Sindbis virus subgenomic	BHK-21	Suspension culture: 0.5 million cells/ml	240,000 c/c	–	(Tate et al. 2003)
Serotonin transporter	β-globin	MEL	Suspension culture: 1 million cells/ml	80,000 c/c	–	(Tate et al. 2003)
Serotonin transporter	CMV/TetO2(pcDNA4)	T-REx 293	–	400,000 c/c	–	(Tate et al. 2003)
Chemokine receptor CCR1	CMV/TetO2(pcDNA4)	T-REx 293	–	Relative fluorescence		(Allen et al. 2009)
Olfactory receptor 17-4	CMV/TetO2(pcDNA4)	HEK293S-TetR	50 × 15 cm plates	∼ 30 ug/15 cm plate	0.13 mg (50 plates)	(Cook et al. 2008)
Olfactory receptor 17-4	CMV/TetO2(pcDNA4)	HEK293S-GnTI^-	Bioreactor (1.25 l): 9.6 million cells/ml	-	3 mg per litre	(Cook et al. 2009)
GABAA receptor α1β3	CMV/TetO2(pcDNA4)	HEK293S-TetR	Bioreactor 3 l	3.2 nmol/l; 14 pmol/mg	1.4 mg	(Dostalova et al. 2010)
Serotonin receptor 5HT3A	CMV/TetO2(pcDNA4)	HEK293S-TetR	Bioreactor 3 l	9–34 nmol/l; 24–47 pmol/mg	5.5 mg	(Dostalova et al. 2010)
Serotonin transporter	CMV/TetO2(pcDNA4)	T-REx 293	3.9 × 10^8 cells from tissue culture plates	1.22–1.67 million c/c	0.59 nmol (∼50 ug)	(Takayama and Sugio 2011)
Vomeronasal receptor 1	CMV/TetO2(pcDNA4)	HEK293S-GnTI^-	Tissue culture plates (unknown quantity)	n.r	1 mg/g of cells	(Corin et al. 2011)
Chemokine receptor CCR1	CMV/TetO2	Flp-In T-REx 293	–	Relative fluorescence only	–	(Allen et al. 2009)
Rhodopsin	CMV/TetO2(pACMV)	HEK293S-TetR	Bioreactor (5.5 l): 5 million cells/ml	9 mg/l	–	(Reeves et al. 2002b)
Rhodopsin	CMV/TetO2(pACMV)	HEK293S-GnTI^-	Bioreactor (1.1 l): 4 million cells/ml	6 mg/l	–	(Reeves et al. 2002a)
β2 adrenergic receptor	CMV/TetO2(pACMV)	HEK293S-TetR	Tissue culture plates	220 pmol/mg mp	12 ug/15 cm plate	(Chelikani et al. 2006)
Bradykinin receptor B2	CMV/TetO2(pACMV)	HEK293S-TetR	–	100 pmol/mg mp; 5.5 million c/c		(Camponova et al. 2007)
Chemokine receptor CCR1	CMV/TetO2(pACMV)	HEK293S-TetR	CellStack or spinner flasks	n.r.	0.1–0.2 mg/ 10^9 cells	(Allen et al. 2009)
Tetraspanin CD81	CMV/TetO2(pACMV)	HEK293S-TetR	15 cm tissue culture plates	∼ 26 ug/30 million cells	–	(Takayama et al. 2008)
Glucagon receptor	CMV/TetO2(pACMV)	HEK293S-GnTI^-	10 cm plates	1.2 ug/mg mp	–	(Unson 2008)
Rhodopsin mutants	CMV/TetO2(pACMV)	HEK293S-GnTI^-	Wave bioreactor 10 l	n.r	∼0.5 mg per litre**	(Standfuss et al. 2011, Deupi et al. 2012)
Ammonia transporter RhCG	CMV/TetO2(pACMV)	HEK293S-GnTI^-	Wave bioreactor: 1 million cells/ml	n.r.	0.5 mg per litre	(Gruswitz et al. 2010, Chaudhary et al. 2012)

*Shown are some examples of membrane proteins expressed in mammalian cells at high-levels or where there are interesting comparisons between expression systems. Further examples can be found in (Grisshammer and Tate 1995, Sarramegna et al. 2003). Expression levels are shown in units as supplied in the primary publication: c/c, copies per cell; mg mp, milligram of membrane protein; n.r., not reported.

**Personal communication.

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