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, 739-745, 2000 Okada et al., PNAS 99, 5982-5987, 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 ([ 3 H]NT) 3-5 nmol/L of culture (0.3 - 0.5 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: 17-39 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: 17-34 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. P950 air sample Ni-NTA AB air frac NiE NiA waste NiB 35% NT0 NT200 NTE NT NT1K 1 2 3 2 4 F3 56 B22 White et al., 2004

20. Large scale purification

21. 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