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

Jim F. White1, Reinhard Grisshammer1

1 NIDDK, 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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Table of Contents

  • 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 ( protocol 3)
  • 2× solubilization buffer (100 mM Tris⋅Cl, pH 7.4/60% v/v glycerol/400 mM NaCl)
  • 1 M MgCl 2
  • 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‐β‐D‐maltoside (LM; Anatrace) detergent stock solution in H 2O; 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 H 2O
    • 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‐β‐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 NaN 3
  • XK50 column (GE Healthcare) packed with 100 ml of Ni‐NTA resin (Qiagen)
  • NT column ( protocol 4)
  • 95% (v/v) ethanol
  • Clarified preparation containing solubilized proteins ( 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 DH5α containing the plasmid pRG/III‐hs‐MBP‐rT43NTR‐TrxA‐H10 (plasmid # 624; see Grisshammer and Tucker, )
  • 1 M isopropyl‐β‐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‐βAla‐βAla‐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‐β‐D‐maltoside (LM; Anatrace)
  • Solubilized NT receptor ( 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‐β‐D‐maltoside (LM; Anatrace) detergent stock solution in H 2O; 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/H 2O
  • Wash solution: 90:2:8 (v/v/v) methanol /acetic acid/ H 2O
  • 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

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Literature Cited

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