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Automated Large‐Scale Purification of a Recombinant G‐Protein‐Coupled Neurotensin Receptor

Jim F. White¹, Reinhard Grisshammer¹

¹NIDDK, NIH, Bethesda, Maryland

Publication Name:

Current Protocols in Protein Science

Unit Number:

Unit 6.8

DOI:

10.1002/0471140864.ps0608s47

Online Posting Date:

February, 2007

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Abstract

Structure determination of G-protein-coupled receptors and other applications, such as nuclear magnetic resonance (NMR) studies, require milligram quantities of purified, functional receptor protein on a regular basis. This unit presents a step-by-step procedure for the automated two-column purification at the 10-milligram scale of a G protein-coupled receptor for neurotensin, expressed in functional form in Escherichia coli.

Keywords: integral membrane proteins; GPCR; detergent; solubilization; purification; automation; ligand binding analysis; protein concentration determination; neurotensin

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Unit Introduction
Basic Protocol 1: Solubilization of Membrane-Bound Receptors from Total Cell Lysate
Basic Protocol 2: Automated Purification of the Nts1 Fusion Protein Using Ni-NTA Chromatography and Neurotensin (NT) Affinity Columns
Support Protocol 1: Expression of Neurotensin Receptor in E. coli
Support Protocol 2: Preparation of the Neurotensin Affinity Column
Support Protocol 3: Ligand-Binding Assay Using Solubilized Receptors
Support Protocol 4: Amido Black Assay for Protein Concentration Determination
Commentary
Appendix: Unicorn 4.11 Programs for Operating The ÄKTA Purifier
Literature Cited
Figures
Tables

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Materials

Basic Protocol 1: Solubilization of Membrane-Bound Receptors from Total Cell Lysate

Materials

250 g frozen E. coli cells expressing the NTS1 fusion protein NTS1-624 (Support Protocol 1)
2× solubilization buffer (100 mM Tris×Cl, pH 7.4/60% v/v glycerol/400 mM NaCl)
1 M MgCl₂
10 mg/ml DNase I (e.g., Sigma)
Deionized water, 4°C
CHAPS/CHS stock solution: 6% (w/v) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS; Anatrace)/1.2% (w/v) cholesteryl hemisuccinate Tris salt (CHS; Anatrace) detergent stock solution; dissolve CHS in CHAPS solution by stirring for several hours; prepare fresh and cool to 4°C
LM stock solution: 10% (w/v) n-dodecyl--d-maltoside (LM; Anatrace) detergent stock solution in H₂O; prepare fresh and cool to 4°C
Protease inhibitor stock solutions
- 70 mg/ml phenylmethylsulfonyl fluoride (PMSF) in ethanol
- 1 mg/ml leupeptin (MP Biomedicals) in H₂O
- 1.4 mg/ml pepstatin A (MP Biomedicals) in methanol
2 M imidazole (e.g., Fluka Ultra; Sigma-Aldrich) stock solution: adjust to pH 7.4 with 12 N HCl

Waring blender
2-liter beaker
Magnetic stir bar and stir plate
Misonix sonicator 3000 with 1/2-inch flat tip
70-ml polycarbonate ultracentrifuge bottles
45Ti ultracentrifugation rotor (Beckman-Coulter; or equivalent) and ultracentrifuge
0.22-µm filter (Stericup; Millipore, or equivalent)

Basic Protocol 2: Automated Purification of the Nts1 Fusion Protein Using Ni-NTA Chromatography and Neurotensin (NT) Affinity Columns

Materials

Milli-Q-purified water (or equivalent)
NiA buffer: 50 mM Tris·Cl, pH 7.4/30% (v/v) glycerol/50 mM imidazole/200 mM NaCl/0.5% (w/v) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS; Anatrace)/0.1% (w/v) cholesteryl hemisuccinate Tris salt (CHS; Anatrace)/0.1% (w/v) n-dodecyl--d-maltoside (LM; Anatrace)
NiB buffer: NiA buffer with 200 mM imidazole
NT0 buffer: 50 mM Tris×Cl, pH 7.4/30% v/v glycerol/1 mM EDTA/0.5% (w/v) CHAPS/0.1% (w/v) CHS/0.1% (w/v) LM
NT200 buffer: NT0 buffer with 200 mM NaCl
NT1K buffer: NT0 buffer with 1 M NaCl
A3 buffer: 50 mM Tris×Cl, pH 7.4/1 mM EDTA/1 M NaCl
B3 buffer: 50 mM Tris×Cl, pH 7.4/1 mM EDTA/3 mM NaN₃
XK50 column (GE Healthcare) packed with 100 ml of Ni-NTA resin (Qiagen)
NT column (Support Protocol 2)
95% (v/v) ethanol
Clarified preparation containing solubilized proteins (Basic Protocol 1)

0.22-µm filter (Stericup; Millipore, or equivalent)
Äkta Purifier P100 chromatography system and operating software, e.g., UNICORN 4.11 (GE Healthcare)
50-ml Falcon test tube
75 borosilicate glass fraction collector tubes (12 × 75 mm)
500- and 1000-ml beakers
250-ml Pyrex bottle

NOTE: All steps are performed at 4°C or on ice unless otherwise indicated. The chromatography equipment should be located in a cold (4°C) room.

Support Protocol 1: Expression of Neurotensin Receptor in E. coli

Materials

2× TY medium (appendix 4A; 200 liters)
100 mg/ml ampicillin stock solution (140 ml)
40% (w/v) glucose stock solution (1 liter)
200-ml overnight 37°C culture of E. coli DH5 containing the plasmid pRG/III-hs-MBP-rT43NTR-TrxA-H10 (plasmid # 624; see Grisshammer and Tucker, 1997)
1 M isopropyl--d-thiogalactopyranoside (IPTG; 50 ml)

300-liter working volume fermentor (New Brunswick Scientific)
Continuous flow centrifuge (Sharples As 16P)

Support Protocol 2: Preparation of the Neurotensin Affinity Column

Materials

Tetrameric avidin resin suspension (Sigma)
50 mM borate buffer, pH 9.0
2 mg/ml succinic anhydride in 50 mM borate buffer, pH 9.0 (dissolves upon gentle warming)
Buffer I (10 mM sodium phosphate pH 7.0)
Buffer II (50 mM Tris×Cl, pH 7.4/1 mM EDTA)
Biotinylated NT (biotin-Ala-Ala-Gln-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu-OH), custom synthesis (a crude preparation may contain some nonbiotinylated NT)
Buffer II containing 5 mM biotin
Buffer II containing 500 mM NaCl
Buffer II containing 3 mM azide
Suitable column (e.g., 2.5 × 10 cm EconoColumn; Bio-Rad)

NOTE: All steps are performed at room temperature.

Support Protocol 3: Ligand-Binding Assay Using Solubilized Receptors

Materials

Receptor dilution buffer: 50 mM Tris×Cl, pH 7.4/1 mM EDTA/0.2% (w/v) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS; Anatrace)/0.04% (w/v) cholesteryl hemisuccinate Tris salt (CHS, Anatrace)/0.1% (w/v) n-dodecyl--d-maltoside (LM; Anatrace)
Solubilized NT receptor (Basic Protocol 2)
4 nM [³H]NT (Perkin-Elmer; specific activity ~100 Ci/mmol or 222,000 dpm/pmol) in TEBB buffer
CHAPS/CHS stock solution: 6% (w/v) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS; Anatrace)/1.2% (w/v) cholesteryl hemisuccinate Tris salt (CHS; Anatrace) detergent stock solution; dissolve CHS in CHAPS solution by stirring for several hours; prepare fresh and cool to 4°C
LM stock solution: 10% (w/v) n-dodecyl--d-maltoside (LM; Anatrace) detergent stock solution in H₂O; prepare fresh and cool to 4°C
TEBB buffer: 50 mM Tris×Cl, pH 7.4/1 mM EDTA /40 µg/ml bacitracin /0.1% (w/v) BSA
40 µM unlabeled neurotensin (NT) solution in TEBB buffer
Scintillant (e.g., Ecoscint A; National Diagnostics)

Bio-Spin 30 Tris columns (Bio-Rad)
Scintillation counter vials (e.g., SnapCap Bio-vials; Beckman),
Centrifuge with swinging-bucket rotor
Liquid scintillation counter

Support Protocol 4: Amido Black Assay for Protein Concentration Determination

Materials

2 mg/ml BSA (Pierce Perbio, albumin standard, fraction V, no. 23209) stock solution
Samples
Tris-SDS solution: 1 M Tris×Cl, pH 7.5/2% (w/v) SDS
6% and 90% (w/w) trichloroacetic acid (TCA)
0.1% (w/v) Amido Black (Sigma; also called Naphthol Blue Black, Amido Black 10B, Buffalo Black NBR) solution in 45:10:45 (v/v/v) methanol/acetic acid/H₂O
Wash solution: 90:2:8 (v/v/v) methanol /acetic acid/ H₂O
Elution solution: 25 mM NaOH, 0.05 mM EDTA in 50% (v/v) ethanol

Custom-made cylindrical Teflon block (one piece with the base 57 mm in diameter and 5 mm high; the upper part 35 mm in diameter and 27 mm high) with 12 evenly spaced holes of 3.5 mm in diameter, to fit on the filtration base
0.45-µm Millipore HA filters (HAWP 047 00)
Millipore glass filtration base with stopper (XX10 047 02) for 47-mm disc filters and anodized aluminum spring clamp (XX10 047 03)
Spectrophotometer with 630-nm capability
1.5-ml disposable plastic cuvettes

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Figures

Figure 6.8.1

Schematic representation of the chromatography system used for automated purification of an NTS1 fusion protein. Circles denote valves (1, injection valve; 2, column valves; 3, outlet valve; 4, sample valve; 5, buffer valve A1; 6, buffer valve B2). The system pumps are indicated as A and B. Buffer lines F3 and B22 are used to collect and to load the Ni-NTA column eluate (NiE) onto the NT column. The NT column flowthrough is collected by line F5. The NT column eluate (NTE) is collected by the fraction collector (frac). Labels in other shaded boxes indicate names of buffers. For clarity, not all buffers and buffer lines are indicated. S1, S2, and S8 represent sample inlet lines. The NiE collected in vessel F3 is diluted with NT0 buffer to reduce the NaCl concentration from 200 mM to 70 mM before passing onto the NT column (syntax setting “35% B” in the UNICORN 4.11 program for purification). Adapted and reprinted by permission of the Federation of the European Biochemical Societies from White et al., 2004.

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Figure 6.8.2

SDS-PAGE of NTS1 fusion protein purification fractions. A 10-mg preparation of the fusion protein NTS1-624 was purified on a 100-ml Ni-NTA column followed by a 20-ml NT column, starting from 250 g of cells. The progress of purification was monitored by SDS-PAGE (NuPAGE 4% to 12% Bis-Tris gel, Invitrogen; 1× MES buffer) and Coomassie R-250 staining. Lane M: Novagen Perfect Protein Marker (15 to 150 kDa); lane 1: 10 µg supernatant; lane 2: 10 µg Ni-NTA column flowthrough; lane 3: 5 µg Ni-NTA column eluate; lane 4: 10 µg NT column flowthrough; lane 5: 5 µg NT column eluate. Reprinted by permission of the Federation of the European Biochemical Societies from White et al., 2004.

View Image

Literature Cited

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