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Large-scale expression and automated purification of G-protein-coupled receptors for structure determination

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

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Large-scale expression and automated purification of G-protein-coupled receptors for structure determination

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  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, 2000Okada et al., PNAS 99, 5982-5987, 2002

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

  4. 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

  5. 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

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

  7. Expression as maltose-binding protein fusion

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

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

  10. Expression levels of NTR fusion protein • 1000 receptors/cell ([3H]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

  11. 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)

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

  13. 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

  14. IMAC Ni-NTA, crude membranes

  15. 1 P950 2 4 NT Ni-NTA air A B 2 NiB sample 5 NiA 6 NT200 NT1K waste 3 NT0 35% frac F3 B22 air NTE NiE White et al., 2004

  16. Large scale purification

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

  18. Generation of T43NTR MBP T43NTR Tev TrxA-H10

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

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