pPIC9K-Msp

Protocol tips

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	The linear recombinant expression plasmid was cut by SalI, and was transformed electronically into GS115. Transformation solution (200 µL) was applied on a MD plate without histidine, and cultured for 3 days at 30°C to screen positive colonies. Single colonies were picked up using sterile pipet tips from the MD plate and placed into YPD media.

Protocol tips
The linear recombinant expression plasmid was cut by SalI, and was transformed electronically into GS115. Transformation solution (200 µL) was applied on a MD plate without histidine, and cultured for 3 days at 30°C to screen positive colonies. Single colonies were picked up using sterile pipet tips from the MD plate and placed into YPD media.

Publication protocol

"P. pastoris competent cells GS115 was prepared following the manufacturer’s guidelines (Invitrogen). The linear recombinant expression plasmid was cut by SalI, and was transformed electronically into GS115. Transformation solution (200 µL) was applied on a MD plate without histidine, and cultured for 3 days at 30°C to screen positive colonies. Single colonies were picked up using sterile pipet tips from the MD plate and placed into YPD media. PCR was performed for colony identification using 1µL lysed supernatant as template. Primers for 5'AOX1 and 3'AOX1 were used to screen for successfully transformed cells. Sterile water was used as blank control.
Positive yeast transformants, which were successfully transformed with pPIC9K-Msp and the empty vector pPIC9K, were inoculated into a 250 mL bottle containing 50 mL BMGY growth media. Yeast was cultured at 30°C and 250 r/min. They were then collected and induced at 28°C for 4 days in 100 mL BMMY media. During induction, samples were collected every 24 h and methanol was added to maintain the anhydrous methanol level at 5 mL/L. Samples were centrifuged at 12,000 g for 2 min. Supernatants were concentrated 10 folds and gene expression was verified by Tricine-SDS PAGE and western blotting as described previously (Zhang et al. 2014)."

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