pSVA1470

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	The methylated expression plasmid pSVA1470 was used for the transformation of electrocompetent MW001 cells as described above. A 400-ml preculture was grown for 3 days in Brock medium containing 0.1% tryptone, 0.2% maltose, and 0.2% sucrose. The whole preculture was used to start fed-batch fermentation in a 10-liter fermentor with continuous air intake.

Protocol tips
The methylated expression plasmid pSVA1470 was used for the transformation of electrocompetent MW001 cells as described above. A 400-ml preculture was grown for 3 days in Brock medium containing 0.1% tryptone, 0.2% maltose, and 0.2% sucrose. The whole preculture was used to start fed-batch fermentation in a 10-liter fermentor with continuous air intake.

Publication protocol

The methylated expression plasmid pSVA1470 was used for the transformation of electrocompetent MW001 cells as described above. A 400-ml preculture was grown for 3 days in Brock medium containing 0.1% tryptone, 0.2% maltose, and 0.2% sucrose. The whole preculture was used to start fed-batch fermentation in a 10-liter fermentor with continuous air intake. The culture was stirred at 200 rpm, and twice a day, fresh medium, including a 0.2% maltose-dextrin mixture as an inducer, was fed. The culture was harvested at an OD600 of 4.1. The harvested cells were disrupted by sonication in buffer A (50 mM Tris-HCl [pH 8.0] and 500 mM NaCl). Cell debris was removed by centrifugation at 25,000 × g, and soluble protein was precipitated with 80% saturated ammonium sulfate. The precipitated protein was resuspended in buffer A supplied with 7.5 mM imidazole, and precipitates were removed by centrifugation at 236,400 × g. The supernatant was loaded onto a 1-ml nickel–nitrilotriacetic acid–agarose column, which was preequilibrated with buffer A containing 7.5 mM imidazole. The column was washed with buffer A containing 50 mM imidazole and was eluted with elution buffer containing 300 mM imidazole. The eluted fraction was pooled and was dialyzed against buffer B (50 mM Tris-HCl [pH 8.0] and 100 mM NaCl) to remove imidazole.

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