pGEX-FIP-nha

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	The plasmid pGEX-FIP-nha was transformed into E. coli Rosetta (Novagen, CA, USA) competent cells. Transformants were grown at 25 °C for 6 h with shaking at 250 rpm in 200 mL of LB medium containing 0.1 mM IPTG and 50 µg/mL ampicillin.	Cells were harvested by centrifugation at 5000× g for 5 min, suspended in 10 mM PBS (10 mL, pH 7.2), and disrupted by sonication. The crude extract was applied to a GST column (Pharmacia, Uppsala, Sweden), equilibrated and eluted with PBS to remove contaminants.

Protocol tips
The plasmid pGEX-FIP-nha was transformed into E. coli Rosetta (Novagen, CA, USA) competent cells. Transformants were grown at 25 °C for 6 h with shaking at 250 rpm in 200 mL of LB medium containing 0.1 mM IPTG and 50 µg/mL ampicillin.
Downstream tips
Cells were harvested by centrifugation at 5000× g for 5 min, suspended in 10 mM PBS (10 mL, pH 7.2), and disrupted by sonication. The crude extract was applied to a GST column (Pharmacia, Uppsala, Sweden), equilibrated and eluted with PBS to remove contaminants.

Publication protocol

The plasmid pGEX-FIP-nha was transformed into E. coli Rosetta (Novagen, CA, USA) competent cells. Transformants were grown at 25 °C for 6 h with shaking at 250 rpm in 200 mL of LB medium containing 0.1 mM IPTG and 50 µg/mL ampicillin. Cells were harvested by centrifugation at 5000× g for 5 min, suspended in 10 mM PBS (10 mL, pH 7.2), and disrupted by sonication. The crude extract was applied to a GST column (Pharmacia, Uppsala, Sweden), equilibrated and eluted with PBS to remove contaminants. Bound protein was cleaved with thrombin (100 U/mL) at 30 °C for 24 h on the column, and cleaved FIP was finally eluted with PBS. Remaining un-cleaved fusion protein and GST were eluted with 10 mM glutathione. Thrombin was removed with Benzamidine sepharose 6B (GE Healthcare, Fairfield, CT, USA). Protein samples were quantified using a NanoDrop-1000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and evaluated with Tricine-SDS-PAGE. In order to exclude the possibility of endotoxin contamination, rFIP-nha was examined by the Limulus Amoebocyte Lysate assay (LAL) (Associates of Cape Cod Inc., East Falmouth, MA, USA), employing the gel clot method (Supplementary Table S1).

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