pPICZαC-MRP4-his6

Protocol tips

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	followed by ligation of MRP4-his6 into the pPICZαC plasmid, at a plasmid-to-insert molar ratio of 1:3, overnight at 16 °C. pPICZαC MRP4-his6 was linearized using PmeI and transformed into P. pastoris ×33 using electroporation.

Protocol tips
followed by ligation of MRP4-his6 into the pPICZαC plasmid, at a plasmid-to-insert molar ratio of 1:3, overnight at 16 °C. pPICZαC MRP4-his6 was linearized using PmeI and transformed into P. pastoris ×33 using electroporation.

Publication protocol

The recombinant pPICZαC-MRP4-his6 construct was created using a double digest of the pFastBac MRP4-his6 plasmid and pPICZαC with EcoRI, followed by ligation of MRP4-his6 into the pPICZαC plasmid, at a plasmid-to-insert molar ratio of 1:3, overnight at 16 °C. pPICZαC MRP4-his6 was linearized using PmeI and transformed into P. pastoris ×33 using electroporation. Colonies containing integrated MRP4 were grown essentially as described previously for Pichia expression of a membrane protein.22 Briefly, colonies were grown in 25 mL of BMGY in sterile 250 mL flasks at 30 °C in a shaking incubator (250–300 rpm) until the culture reached an OD600 of 2–6. Cells were harvested by centrifugation at 3000g for 5 min, all BMGY was removed, and then they were washed with BMMY and resuspended in BMMY at an OD600 of 1.0 before being returned to the shaking incubator at 22 or 30 °C. Sterilized pure methanol was added every 24 h to a final concentration of 0.5% (v/v) methanol. Samples were taken every 24 h over a 72 h period.

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