1. Large-scale expression and automated purification of G-protein-coupled receptors for structure determination
Jim F. White 1, Loc B. Trinh 2,
Joseph Shiloach 2 and Reinhard Grisshammer 1
1 Laboratory of Molecular Biology
2 Biotechnology Unit
NIDDK, NIH, Bethesda MD
Department of Health and Human Services

2. G-protein-coupled receptors
Palczewski et al., Science 289, , 2000 Okada et al., PNAS 99, , 2002

4. Structure determination of membrane receptors
Gene or cDNA isolation
System for high level expression
Solubilization and purification
Characterization and crystallization
Structure determination

5. Rat neurotensin receptor (NTR, NTS-1)
424 aa, 47 kDa
rat brain cDNA library
Tanaka et al., Neuron 4, 847, 1990
cDNA from S. Nakanishi
neurotensin
Glp-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu

6. Expression of seven-helix G-protein coupled receptors in Escherichia coli
Practical aspects
maintenance of “cell line” is easy
scale-up of expression is possible
cell breakage at large scale not problematic

8. Tools for assessing expression levels
functional receptors → ligand binding analysis
total receptor protein → Western blot (‘tag’)

10. Expression as maltose-binding protein fusion

12. Influence of tag on expression
Tucker & Grisshammer, 1996

13. Expression in E. coli of the neurotensin receptor fusion protein

14. Expression levels of NTR fusion protein
1000 receptors/cell ([3H]NT)
3-5 nmol/L of culture ( mg/L)
9 pmol/mg of total solubilized protein
24 pmol/mg of membrane protein

15. General applicability of expression system
Cannabinoid CB1 receptor: No (degraded)
Cannabinoid CB2 receptor: pmol/mg
(MBP-CB2-HF) (Calandra et al., 1997)
Substance K receptor: 7 pmol/mg
(MBP-SKR-HMTX) (Grisshammer et al., 1994)
Neurotensin receptor: 24 pmol/mg
(MBP-T43NTR-TrxA-H10) (Grisshammer & Tucker, 1997)
Adenosine A2a receptor: pmol/mg
(MBP-A2aTr316-H10) (Weiß & Grisshammer, 2002)

16. Purification of the neurotensin receptor fusion protein
Ni-NTA 1.
IMAC 2.
ligand affinity column

17. Immobilized metal affinity chromatography (IMAC)
His tag HHHHHHHHHH
very robust purification system
easy to scale up
excellent purification of NTR fusion protein from total cell lysate
Grisshammer & Tucker, 1997

18. IMAC Ni-NTA, crude membranes

19. NT 1 P950 2 4 Ni-NTA air A B 2 NiB sample 5 NiA 6 NT200 NT1K waste 3
35%
frac F3
B22
air
NTE
NiE
White et al., 2004

20. Large scale purification

21. Large scale purification
1: SN
2: NiFT
3: NiE
4: NTFT
5: NTE

22. Generation of T43NTR
MBP
T43NTR
Tev
TrxA-H10

23. Backbone conformation of NT(8-13) bound to rat NTS-1
Luca et al., 2003