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Acknowledgement Structural Bioinformatics Laboratory PowerPoint Presentation

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  • Slide 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
  • Slide 2 - G-protein-coupled receptors Palczewski et al., Science 289, 739-745, 2000 Okada et al., PNAS 99, 5982-5987, 2002
  • Slide 3 - ppt slide no 3 content not found
  • Slide 4 - Structure determination of membrane receptors Gene or cDNA isolation System for high level expression Solubilization and purification Characterization and crystallization Structure determination
  • Slide 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
  • Slide 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
  • Slide 7 - ppt slide no 7 content not found
  • Slide 8 - Tools for assessing expression levels functional receptors → ligand binding analysis total receptor protein → Western blot (‘tag’)
  • Slide 9 - ppt slide no 9 content not found
  • Slide 10 - Expression as maltose-binding protein fusion
  • Slide 11 - ppt slide no 11 content not found
  • Slide 12 - Influence of tag on expression Tucker & Grisshammer, 1996
  • Slide 13 - Expression in E. coli of the neurotensin receptor fusion protein
  • Slide 14 - 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
  • Slide 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)
  • Slide 16 - Purification of the neurotensin receptor fusion protein Ni-NTA 1. IMAC 2. ligand affinity column
  • Slide 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
  • Slide 18 - IMAC Ni-NTA, crude membranes
  • Slide 19 - P950 air sample Ni-NTA A B air frac NiE NiA waste NiB 35% NT0 NT200 NTE NT NT1K 1 2 3 2 4 F3 5 6 B22 White et al., 2004
  • Slide 20 - Large scale purification
  • Slide 21 - Large scale purification 1: SN 2: NiFT 3: NiE 4: NTFT 5: NTE
  • Slide 22 - Generation of T43NTR MBP T43NTR Tev TrxA-H10
  • Slide 23 - Backbone conformation of NT(8-13) bound to rat NTS-1 Luca et al., 2003

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