pFastBac1-TEV-TRPV1

Protocol tips

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	For protein expression, HEK293S GnTI− cells46, grown in suspension at 37 °C in an orbital shaker, were transduced when cell density reached ∼ 2 × 106 per ml. Sodium butyrate was added to the culture 24 h after transduction at a final concentration of 10 mM to boost protein expression, and cells collected 48 h after transduction for preparation of crude membrane and subsequent protein purification, as described10 with slight modification.

Protocol tips

For protein expression, HEK293S GnTI− cells46, grown in suspension at 37 °C in an orbital shaker, were transduced when cell density reached ∼ 2 × 106 per ml. Sodium butyrate was added to the culture 24 h after transduction at a final concentration of 10 mM to boost protein expression, and cells collected 48 h after transduction for preparation of crude membrane and subsequent protein purification, as described10 with slight modification.

Publication protocol

"A BacMam vector was generated from pFastBac1 (Invitrogen) to direct protein expression in mammalian cells after baculovirus transduction45. In brief, the polyhedron promoter (PPH) in pFastBac1 was replaced with a mammalian cell active promoter (PCMV), immediately followed by an N-terminal fusion cassette (Kozac-MBP-tobacco etch virus (TEV) protease site) for affinity purification with amylose resin (New England Biolabs). A minimal functional rat TRPV1 construct, composed of amino acids 110 to 603 and 627 to 764, was cloned into this modified BacMam vector, and recombinant baculoviruses obtained following the manufacturer’s protocol (Bac-to-Bac expression system, Invitrogen). For protein expression, HEK293S GnTI− cells46, grown in suspension at 37 °C in an orbital shaker, were transduced when cell density reached ∼ 2 × 106 per ml. Sodium butyrate was added to the culture 24 h after transduction at a final concentration of 10 mM to boost protein expression, and cells collected 48 h after transduction for preparation of crude membrane and subsequent protein purification, as described10 with slight modification. TRPV1 channel was eluted from amylose resin with buffer composed of 150 mM NaCl, 2 mM tris(2-carboxyethyl)phosphine (TCEP), 10% glycerol, 20 mM HEPES, 0.5 mM DDM, 0.1 mg ml−1 soybean lipids, and 20 mM maltose, then incubated with TEV protease for 4 h at 4 °C. The cleaved protein sample was then mixed with amphipols at 1:3 (w/w) with gentle agitation for another 4 h. Detergent was removed with Bio-Beads SM-2 (4 °C overnight, 15 mg per 1 ml channel/detergent/amphipols mixture). Bio-beads were then removed over a disposable polyprep column, and eluent cleared by centrifugation before further separation on a Superdex 200 column in buffer composed of 150 mM NaCl, 20 mM HEPES, 2 mM TCEP, pH 7.4. The peak corresponding to tetrameric TRPV1 channels was collected for analysis by cryo-EM.

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Download the product protocol from Yifan Cheng, Department of Biochemistry and Biophysics, Keck Adv for pFastBac1-TEV-TRPV1 below.

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