pHUE -Ls-AChBP

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	The pHUE expression construct for Ls-AChBP transformed into BL21DE3 cells were used for the expression. Cells were grown in LB broth to an O.D of 0.7–0.8 after which they were pelleted at 5,000 x g for 30 min.

Protocol tips
The pHUE expression construct for Ls-AChBP transformed into BL21DE3 cells were used for the expression. Cells were grown in LB broth to an O.D of 0.7–0.8 after which they were pelleted at 5,000 x g for 30 min.

Publication protocol

The protein expression method followed here is adapted from Marley et al., 2010 and Shivashanmugam et al. [40, 41]. The pHUE expression construct for Ls-AChBP transformed into BL21DE3 cells were used for the expression. Cells were grown in LB broth to an O.D of 0.7–0.8 after which they were pelleted at 5,000 x g for 30 min. The pellets were gently re-suspended in 1 x M9 salts (22 mM KH2PO4, 90 mM Na2HPO4, 17 mM NaCl) to remove residual LB broth. The re-suspended cells were again pelleted at 5,000 x g for 30 min, re-suspended in 1L of minimal media (1 x M9 salts 20% (w/v),Vitamin solution 0.2% (w/v),Thiamine solution 0.2% (w/v),1 M MgSO4 0.16% (w/v), 1 M CaCl2 0.008% (w/v), 15NH4Cl 0.1% (w/v), 13C D-glucose 0.4% (w/v), Ampicillin 0.1 mg/mL) and grown at 37°C for 1–1.5 hours before protein expression was induced with 1 mM IPTG (final concentration) at 16°C for 18–20 h. Finally, cells were harvested at 5,000 x g for 30 min at 4°C, the pellet re-suspended in 20 mM Tris, 150 mM NaCl, 10% glycerol and pH 8.0 and stored at –80°C prior to use.

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