Cloning, expression, and purification of Twitch-2B and Twitch-6
This protocol is extracted from research article:
Dynamic tuning of FRET in a green fluorescent protein biosensor
Sci Adv, Aug 7, 2019; DOI: 10.1126/sciadv.aaw4988

The Twitch-2B construct has been described before (8). For the present study, the coding sequence of the three-domain fusion protein was cloned into a modified pET16b vector coding for a fusion protein with N-terminal His7-tag and tabac etch virus (TEV) recognition cleavage sequence. The Twitch-2B N532F mutant (Twitch-6) was generated using the QuikChange site-directed mutagenesis kit (Agilent). The pET16bTEV-Twitch-2B and pET16bTEV-Twitch-6 expression constructs were transformed into Escherichia coli strain BL21 (DE3). Protein expression was carried out at 303 K by induction with 0.5 mM isopropyl β-d-1-thiogalactopyranoside. The cells were harvested 7 hours after induction. Selenomethionine-labeled Twitch-2B protein was overexpressed in the methionine-auxotroph E. coli strain B834 in minimal medium supplemented with (+)-l-selenomethionine according to the EMBL (European Molecular Biology Laboratory) protein-expression group (www.embl.de).

The cell pellet from 1 liter of shaking culture was resuspended in 60 ml of lysis buffer [20 mM tris-HCl (pH 7.9), 300 mM NaCl, 20 mM imidazole, 0.5 mM phenylmethylsulfonyl fluoride, with one tablet of cOmplete EDTA-free inhibitors (Roche) per 100 ml of lysis buffer]. The cells were lysed by sonication followed by centrifugation at 27,000g and 277 K. From the supernatant, the recombinant protein was purified by immobilized metal-affinity chromatography on 3 ml of Ni–nitrilotriacetic acid (NTA) agarose resin (Qiagen). The His7-fusion tag was cleaved off with TEV protease and removed by incubation with 1 ml of Ni-NTA agarose resin. The protein was dialyzed against 20 mM tris (pH 7.0) and 150 mM NaCl. After adjusting the ammonium sulfate concentration in the protein solution to 1 M, the protein was further purified by hydrophobic interaction chromatography on a 10-ml Phenyl Sepharose (GE Healthcare) column. The protein was eluted from this column with a 50-ml gradient from 1 to 0 M ammonium sulfate. Fractions containing the protein were pooled and concentrated to a volume of 2.5 ml with a 30-kDa MWCO (molecular weight cut-off) ultrafiltration concentrator (Vivascience). Last, the protein was purified by size exclusion chromatography on a HiLoad 26/60 Superdex 200-pg gel filtration column. The peak fractions were pooled, dialyzed against 20 mM tris-HCl (pH 7.0), 100 mM NaCl, and 5 mM CaCl2, and the protein concentration was adjusted to 20 mg/ml.

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