pTXB1-MnP1

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	For expression of the fusion protein consisting of recombinant MnP1 (rMnP1), intein, and a chitin binding domain (rMnP1−intein−CBD), five mL of LB, supplemented with 100 µg mL−1 of ampicillin (LBamp), was inoculated with single colonies of E. coli T7 shuffle pTXB1-MnP1 and incubated overnight. Then, 25 mL of LBamp was inoculated with 2 mL of the overnight culture and incubated until the OD600nm reached 0.5. One litre of LBamp was inoculated with the whole 25-mL culture and incubated until OD600nm again reached 0.5. Next, protein expression was induced with the addition of 0.1 mM isopropyl-β-D thiogalactopyranoside (IPTG) and incubated for 12 h.

Protocol tips

For expression of the fusion protein consisting of recombinant MnP1 (rMnP1), intein, and a chitin binding domain (rMnP1−intein−CBD), five mL of LB, supplemented with 100 µg mL−1 of ampicillin (LBamp), was inoculated with single colonies of E. coli T7 shuffle pTXB1-MnP1 and incubated overnight. Then, 25 mL of LBamp was inoculated with 2 mL of the overnight culture and incubated until the OD600nm reached 0.5. One litre of LBamp was inoculated with the whole 25-mL culture and incubated until OD600nm again reached 0.5. Next, protein expression was induced with the addition of 0.1 mM isopropyl-β-D thiogalactopyranoside (IPTG) and incubated for 12 h.

Publication protocol

For expression of the fusion protein consisting of recombinant MnP1 (rMnP1), intein, and a chitin binding domain (rMnP1−intein−CBD), five mL of LB, supplemented with 100 µg mL−1 of ampicillin (LBamp), was inoculated with single colonies of E. coli T7 shuffle pTXB1-MnP1 and incubated overnight. Then, 25 mL of LBamp was inoculated with 2 mL of the overnight culture and incubated until the OD600nm reached 0.5. One litre of LBamp was inoculated with the whole 25-mL culture and incubated until OD600nm again reached 0.5. Next, protein expression was induced with the addition of 0.1 mM isopropyl-β-D thiogalactopyranoside (IPTG) and incubated for 12 h. Induction was tested with 0.4 or 0.8 mM IPTG added after 4, 8, or 24 h of incubation time. Cells were harvested by centrifugation at 3000× g at 4 °C for 30 min. About 6 g of wet cell pellet was recovered from 1 L of culture. From there, 1.7 g of pellet was re-suspended in 15 mL of lysis buffer (20 mM Tris-HCl pH 8.5; 500 mM NaCl; 1 mM EDTA; 0.1% v/v Triton X-100; 20 µM PMSF) and sonicated on ice. The lysate was centrifuged at 15,000× g for 30 min at 4 °C. Both soluble and insoluble fractions were analysed by SDS-PAGE.

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