pET42-cIL18

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	A 1-liter culture of E. coli (BL21 strain) transformed with pET42-cIL18 was incubated at 37°C. When the optical density at 600 nm (OD600) reached 0.4, expression of the recombinant protein was induced by the addition of 1 mM isopropyl-β-D-thiogalactopyranoside. After a 4-hr incubation, the E. coli was washed with PBS, suspended with sonication buffer (0.5% Triton X-100, 50 mM Tris-HCl [pH 8.0], 1 mM EDTA and 10 mM dithiothreitol) and sonicated with Sonifier450 (Branson, North Olmsted, OH, U.S.A.) for 5 min on ice.	The lysate was incubated with 0.2 mg/ml lysozyme (Wako, Osaka, Japan), 10 µg/ml DNase I (Roche, Mannheim, Germany) and 1 mM MgCl2 for 45 min at room temperature. The lysate was added with 7 mM EDTA, incubated for 30 min at 37°C and then centrifugation at 12,000 × g for 20 min.

Protocol tips
A 1-liter culture of E. coli (BL21 strain) transformed with pET42-cIL18 was incubated at 37°C. When the optical density at 600 nm (OD600) reached 0.4, expression of the recombinant protein was induced by the addition of 1 mM isopropyl-β-D-thiogalactopyranoside. After a 4-hr incubation, the E. coli was washed with PBS, suspended with sonication buffer (0.5% Triton X-100, 50 mM Tris-HCl [pH 8.0], 1 mM EDTA and 10 mM dithiothreitol) and sonicated with Sonifier450 (Branson, North Olmsted, OH, U.S.A.) for 5 min on ice.
Downstream tips
The lysate was incubated with 0.2 mg/ml lysozyme (Wako, Osaka, Japan), 10 µg/ml DNase I (Roche, Mannheim, Germany) and 1 mM MgCl2 for 45 min at room temperature. The lysate was added with 7 mM EDTA, incubated for 30 min at 37°C and then centrifugation at 12,000 × g for 20 min.

Publication protocol

The cDNA of cIL-18 was cloned into pET42 (b) E. coli expression vector (Novagen, Darmstadt, Germany) in frame with C-terminal of glutathione-s-transferase (GST) (pET42-cIL18). A 1-liter culture of E. coli (BL21 strain) transformed with pET42-cIL18 was incubated at 37°C. When the optical density at 600 nm (OD600) reached 0.4, expression of the recombinant protein was induced by the addition of 1 mM isopropyl-β-D-thiogalactopyranoside. After a 4-hr incubation, the E. coli was washed with PBS, suspended with sonication buffer (0.5% Triton X-100, 50 mM Tris-HCl [pH 8.0], 1 mM EDTA and 10 mM dithiothreitol) and sonicated with Sonifier450 (Branson, North Olmsted, OH, U.S.A.) for 5 min on ice. The lysate was incubated with 0.2 mg/ml lysozyme (Wako, Osaka, Japan), 10 µg/ml DNase I (Roche, Mannheim, Germany) and 1 mM MgCl2 for 45 min at room temperature. The lysate was added with 7 mM EDTA, incubated for 30 min at 37°C and then centrifugation at 12,000 × g for 20 min.

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