■ TRPV1: 20 mM HEPES, 150 mM NaCl, 2 mM TCEP, 10% glycerol, 0.2 g‧L
À1 soybean
lipids (asolectin), 0.5 mM DDM, pH 8.0.
□ TRPA1: 20 mM HEPES, 150 mM NaCl, 1 mM DTT, 1 mM IP 6 , 0.5 mM Lauryl Maltose
Neopentyl Glycol (LMNG, MNG-3), pH 8.0.
• Elution buffer:
■ TRPV1: 20 mM HEPES, 150 mM NaCl, 2 mM TCEP, 10% glycerol, 0.2 g‧L
À1 asolectin,
0.5 mM DDM, 20 mM maltose, pH 8.0.
□ TRPA1: 20 mM HEPES, 150 mM NaCl, 1 mM DTT, 1 mM IP 6 , 0.5 mM MNG-3, 40 mM
maltose, pH 8.0.
• Size exclusion chromatography buffer:
■ TRPV1: 20 mM HEPES, 150 mM NaCl, 2 mM TCEP, pH 7.4.
□ TRPA1: 20 mM HEPES, 150 mM NaCl, 1 mM DTT, 1 mM IP 6 , pH 8.0.
12.4.4 Protocol
1. Transduce HEK293S GnTi
À cells grown in suspension at 37
C in an orbital shaker at a
density of 1.5–2 Â 10
6 cells per mL with 1:10 (v/v)–1:50 (v/v) baculovirus encoding the
protein of interest.
The volume of baculovirus to use to transduce cells can be readily determined by a Western
blot-based dilution series expression test. Briefly, transduce HEK293T or HEK293S GnTi
À
cells in a 6-well dish with 0, 1:10, 1:20, 1:30, 1:40, or 1:50 (v/v) baculovirus. Add sodium
butyrate at a final concentration of 10 mM. After 16–24 h, collect cells, generate cell lysates,
and use for anti-MBP Western blot. Choose the lowest volume of virus that yields the
maximum protein production.
2. Twenty-four hours after transduction, add sodium butyrate to the culture at a final concentration of 5–10 mM to boost protein expression.
An alternative expression protocol that is commonly used and can serve as a protocol
optimization point is to transduce HEK293S GnTi
À cells with baculovirus and add sodium
butyrate at the same time.
3. Harvest cells 48 h after transduction by centrifugation (3000 rpm, 15 min at 4
C).
The length of transduction time is another protocol optimization point. Typically, cells are
transduced for 30–48 h. When cells are transduced for 30 h, sodium butyrate is added at the
same time as baculovirus. Typically, the cells are transduced in the presence of sodium
butyrate for at least 24 h. Though not routinely done in our lab, growing HEK293 cells at
30
C after adding sodium butyrate can often enhance protein expression (Goehring et al.
2014).
4. Resuspend cell pellets in lysis buffer (50 mL lysis buffer for each liter of cells) and break cells
by passing through an emulsifier twice.
12.4 Protocol 12.1: Preparation of Amphipol-Trapped Membrane Proteins for Cryo-EM Studies
581
À1 soybean
lipids (asolectin), 0.5 mM DDM, pH 8.0.
□ TRPA1: 20 mM HEPES, 150 mM NaCl, 1 mM DTT, 1 mM IP 6 , 0.5 mM Lauryl Maltose
Neopentyl Glycol (LMNG, MNG-3), pH 8.0.
• Elution buffer:
■ TRPV1: 20 mM HEPES, 150 mM NaCl, 2 mM TCEP, 10% glycerol, 0.2 g‧L
À1 asolectin,
0.5 mM DDM, 20 mM maltose, pH 8.0.
□ TRPA1: 20 mM HEPES, 150 mM NaCl, 1 mM DTT, 1 mM IP 6 , 0.5 mM MNG-3, 40 mM
maltose, pH 8.0.
• Size exclusion chromatography buffer:
■ TRPV1: 20 mM HEPES, 150 mM NaCl, 2 mM TCEP, pH 7.4.
□ TRPA1: 20 mM HEPES, 150 mM NaCl, 1 mM DTT, 1 mM IP 6 , pH 8.0.
12.4.4 Protocol
1. Transduce HEK293S GnTi
À cells grown in suspension at 37
C in an orbital shaker at a
density of 1.5–2 Â 10
6 cells per mL with 1:10 (v/v)–1:50 (v/v) baculovirus encoding the
protein of interest.
The volume of baculovirus to use to transduce cells can be readily determined by a Western
blot-based dilution series expression test. Briefly, transduce HEK293T or HEK293S GnTi
À
cells in a 6-well dish with 0, 1:10, 1:20, 1:30, 1:40, or 1:50 (v/v) baculovirus. Add sodium
butyrate at a final concentration of 10 mM. After 16–24 h, collect cells, generate cell lysates,
and use for anti-MBP Western blot. Choose the lowest volume of virus that yields the
maximum protein production.
2. Twenty-four hours after transduction, add sodium butyrate to the culture at a final concentration of 5–10 mM to boost protein expression.
An alternative expression protocol that is commonly used and can serve as a protocol
optimization point is to transduce HEK293S GnTi
À cells with baculovirus and add sodium
butyrate at the same time.
3. Harvest cells 48 h after transduction by centrifugation (3000 rpm, 15 min at 4
C).
The length of transduction time is another protocol optimization point. Typically, cells are
transduced for 30–48 h. When cells are transduced for 30 h, sodium butyrate is added at the
same time as baculovirus. Typically, the cells are transduced in the presence of sodium
butyrate for at least 24 h. Though not routinely done in our lab, growing HEK293 cells at
30
C after adding sodium butyrate can often enhance protein expression (Goehring et al.
2014).
4. Resuspend cell pellets in lysis buffer (50 mL lysis buffer for each liter of cells) and break cells
by passing through an emulsifier twice.
12.4 Protocol 12.1: Preparation of Amphipol-Trapped Membrane Proteins for Cryo-EM Studies
581