pCRM197

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	For protein expression, precultures were prepared by inoculating the recombinant strains in 5 ml LB (10 g l−1 tryptone, 5 g l−1 yeast extract, 10 g l−1 NaCl) medium containing kanamycin (50 mg l−1) and/or chloramphenicol (34 mg l−1), depending on plasmids present, and cultivated overnight at 37°C. A 1 in 100 dilution was made into 50 ml fresh LB media and cultivation continued at 30°C until OD600 = 0·6, followed by induction with 0·5 mol l−1 isopropyl‐β‐d‐thiogalactopyranoside (IPTG).

Protocol tips

For protein expression, precultures were prepared by inoculating the recombinant strains in 5 ml LB (10 g l−1 tryptone, 5 g l−1 yeast extract, 10 g l−1 NaCl) medium containing kanamycin (50 mg l−1) and/or chloramphenicol (34 mg l−1), depending on plasmids present, and cultivated overnight at 37°C. A 1 in 100 dilution was made into 50 ml fresh LB media and cultivation continued at 30°C until OD600 = 0·6, followed by induction with 0·5 mol l−1 isopropyl‐β‐d‐thiogalactopyranoside (IPTG).

Publication protocol

For protein expression, precultures were prepared by inoculating the recombinant strains in 5 ml LB (10 g l−1 tryptone, 5 g l−1 yeast extract, 10 g l−1 NaCl) medium containing kanamycin (50 mg l−1) and/or chloramphenicol (34 mg l−1), depending on plasmids present, and cultivated overnight at 37°C. A 1 in 100 dilution was made into 50 ml fresh LB media and cultivation continued at 30°C until OD600 = 0·6, followed by induction with 0·5 mol l−1 isopropyl‐β‐d‐thiogalactopyranoside (IPTG). The cell pellets were harvested by centrifugation 20 h after induction, then resuspended in an equivalent volume Binding Buffer (20 mmol l−1 Tris pH 7·9, 500 mmol l−1 NaCl, 5 mmol l−1 imidazole) and sonicated for 5 min on continuous pulse using a sonicator (SonoPuls, Bandelin Electronic, Berlin, Germany) at 67% power. Centrifugation for 10 min at 9168 g yielded supernatant (soluble fraction) and pellet (insoluble fraction). The pellet was again resuspended in an equivalent volume Binding Buffer. Soluble and insoluble fractions were visualized on 12% SDS‐PAGE gels.

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