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

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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 ... – PowerPoint PPT presentation

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


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
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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-L
    eu

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

7
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8
Tools for assessing expression levels
  • functional receptors ? ligand binding analysis
  • total receptor protein ? Western blot (tag)

9
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10
Expression as maltose-binding protein fusion
11
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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 (3HNT)
  • 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
1
P950
2
4
NT
Ni-NTA
air
A
B
NiB
2
sample
NiA
5
6
NT200
NT1K
waste
3
NT0
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
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