Fig. 12.20, and additional references given at the end of § 12.3.3). The following is an annotated
protocol for purifying TRPV1 and TRPA1 in detergent solution and transferring them to APols for
structural studies. Experimental steps that are common to both MPs are marked with a black bullet (•)
and details that are specific to TRPV1 and TRPA1 with ■ and □ symbols, respectively; general
comments and tips for optimization are in italics and marked with a pointing hand sign ( ).
12.4.2 Vectors and Cell Lines
• pFastBac1 plasmid containing the gene of interest in frame with an N-terminal maltose
binding protein (MBP) affinity tag and a TEV protease site cloned downstream of an
engineered human cytomegalovirus (CMV) promoter for baculovirus transduction-based
expression in HEK293S GnTi
À cells
MPs can be expressed in a number of expression systems including suspension of HEK cells,
Sf 9 insect cells, yeast, and bacteria. The most appropriate expression system for each protein of
interest needs to be determined empirically. For TRPV1, well-behaved, homogenous protein can
be purified from both suspensions of HEK and Sf 9 cells (Cao et al. 2013a). For TRPA1, wellbehaved, homogeneous protein could only be purified from suspensions of HEK cells.
• HEK293S GnTi
À cells (ATCC, catalogue number CRL-3022)
• Sf 9 cells (Life Technologies, catalogue number 12659017)
Detailed protocols for the growth and maintenance of Sf 9 cells for baculovirus generation
and HEK293S GnTi
À cells for the expression of MPs are given in Goehring et al. (2014).
12.4.3 Buffers
• Lysis buffer:
■ TRPV1: 50 mM Tris/HCl buffer, 36.5 mM sucrose, 2 mM tris(2-carboxyethyl)phosphine
(TCEP), pH 8.0, supplemented with 1 mM phenylmethanesulphonylfluoride (PMSF), 3 mg‧
L
-1 leupeptin, 1 mg‧L
-1 pepstatin.
□ TRPA1: 50 mM Tris/HCl buffer, 36.5 mM sucrose, 1 mM β-mercaptoethanol (β-ME),
5 mM EDTA, 1 mM inositol hexakisphosphate (IP 6 ), pH 8.0, supplemented with 1Â protease
inhibitor cocktail tablet (Roche). 1Â protease inhibitor cocktail is produced by dissolving one
tablet in 50 mL of buffer.
• Membrane resuspension buffer:
■ TRPV1: 50 mM HEPES, 200 mM NaCl, 2 mM TCEP, 10% glycerol, pH 8.0,
supplemented with 1 mM PMSF, 3 mg‧L
À1 leupeptin, 1 mg‧L
À1 pepstatin.
□ TRPA1: 50 mM HEPES, 150 mM NaCl, 1 mM dithiothreitol (DTT), 1 mM IP 6 , 10%
glycerol, pH 8.0, supplemented with 1Â protease inhibitor cocktail tablet (Roche). 1Â
protease inhibitor cocktail is produced by dissolving one tablet in 50 mL of buffer.
• Wash buffer:
580
12 The Use of Amphipols for Electron Microscopy
protocol for purifying TRPV1 and TRPA1 in detergent solution and transferring them to APols for
structural studies. Experimental steps that are common to both MPs are marked with a black bullet (•)
and details that are specific to TRPV1 and TRPA1 with ■ and □ symbols, respectively; general
comments and tips for optimization are in italics and marked with a pointing hand sign ( ).
12.4.2 Vectors and Cell Lines
• pFastBac1 plasmid containing the gene of interest in frame with an N-terminal maltose
binding protein (MBP) affinity tag and a TEV protease site cloned downstream of an
engineered human cytomegalovirus (CMV) promoter for baculovirus transduction-based
expression in HEK293S GnTi
À cells
MPs can be expressed in a number of expression systems including suspension of HEK cells,
Sf 9 insect cells, yeast, and bacteria. The most appropriate expression system for each protein of
interest needs to be determined empirically. For TRPV1, well-behaved, homogenous protein can
be purified from both suspensions of HEK and Sf 9 cells (Cao et al. 2013a). For TRPA1, wellbehaved, homogeneous protein could only be purified from suspensions of HEK cells.
• HEK293S GnTi
À cells (ATCC, catalogue number CRL-3022)
• Sf 9 cells (Life Technologies, catalogue number 12659017)
Detailed protocols for the growth and maintenance of Sf 9 cells for baculovirus generation
and HEK293S GnTi
À cells for the expression of MPs are given in Goehring et al. (2014).
12.4.3 Buffers
• Lysis buffer:
■ TRPV1: 50 mM Tris/HCl buffer, 36.5 mM sucrose, 2 mM tris(2-carboxyethyl)phosphine
(TCEP), pH 8.0, supplemented with 1 mM phenylmethanesulphonylfluoride (PMSF), 3 mg‧
L
-1 leupeptin, 1 mg‧L
-1 pepstatin.
□ TRPA1: 50 mM Tris/HCl buffer, 36.5 mM sucrose, 1 mM β-mercaptoethanol (β-ME),
5 mM EDTA, 1 mM inositol hexakisphosphate (IP 6 ), pH 8.0, supplemented with 1Â protease
inhibitor cocktail tablet (Roche). 1Â protease inhibitor cocktail is produced by dissolving one
tablet in 50 mL of buffer.
• Membrane resuspension buffer:
■ TRPV1: 50 mM HEPES, 200 mM NaCl, 2 mM TCEP, 10% glycerol, pH 8.0,
supplemented with 1 mM PMSF, 3 mg‧L
À1 leupeptin, 1 mg‧L
À1 pepstatin.
□ TRPA1: 50 mM HEPES, 150 mM NaCl, 1 mM dithiothreitol (DTT), 1 mM IP 6 , 10%
glycerol, pH 8.0, supplemented with 1Â protease inhibitor cocktail tablet (Roche). 1Â
protease inhibitor cocktail is produced by dissolving one tablet in 50 mL of buffer.
• Wash buffer:
580
12 The Use of Amphipols for Electron Microscopy