pONE-25A

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	Cells were plated on Minimal Dextrose (MD) plates (for vectors with HIS4 metabolic marker) and incubated for 3 days at 30°C. Freshly grown colonies from the plates were picked and grown in 20 mL or 3×100 mL YPD medium at 30°C overnight at 280 rpm. The pre-inoculum was used to inoculate 200 mL or 3×1 L Buffered Media with Glycerol (BMGY) medium and were grown for 8 hours at 30°C at 280 rpm in Erlenmeyer flasks. The flasks were not filled with more media than 25% of nominal volume.

Protocol tips

Cells were plated on Minimal Dextrose (MD) plates (for vectors with HIS4 metabolic marker) and incubated for 3 days at 30°C. Freshly grown colonies from the plates were picked and grown in 20 mL or 3×100 mL YPD medium at 30°C overnight at 280 rpm. The pre-inoculum was used to inoculate 200 mL or 3×1 L Buffered Media with Glycerol (BMGY) medium and were grown for 8 hours at 30°C at 280 rpm in Erlenmeyer flasks. The flasks were not filled with more media than 25% of nominal volume.

Publication protocol

For expression in P. pastoris, recombinant protein containing vectors were linearized using PmeI or AvrII restriction enzyme–depending on the vector–and transformed via electroporation. Cleaved vector DNA targets the vector for integration into the yeast genome at the cleavage region. Without cleavage transformation efficiency is low and may result in non-expressing clones. PmeI site is present in the vectors with AOX1 promoter, while AvrII is present in the GAP promoter. It should be noted that AvrII is not unique, however the other AvrII site is eliminated in most cloning strategies. If not, the unique BglII site may be used, albeit this results in lower transformation efficiency. Cells were plated on Minimal Dextrose (MD) plates (for vectors with HIS4 metabolic marker) and incubated for 3 days at 30°C. Freshly grown colonies from the plates were picked and grown in 20 mL or 3×100 mL YPD medium at 30°C overnight at 280 rpm. The pre-inoculum was used to inoculate 200 mL or 3×1 L Buffered Media with Glycerol (BMGY) medium and were grown for 8 hours at 30°C at 280 rpm in Erlenmeyer flasks. The flasks were not filled with more media than 25% of nominal volume.

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