pPICZ-B

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	Vector was linearized with Pme1 and transformed into P. pastoris strain SMD1163 by electroporation. Transformants were selected by plating on YPDS plates with 1mg/mL zeocin.

Protocol tips
Vector was linearized with Pme1 and transformed into P. pastoris strain SMD1163 by electroporation. Transformants were selected by plating on YPDS plates with 1mg/mL zeocin.

Publication protocol

"A gene corresponding to H. sapiens TRAAK (GI:13124080) amino acids 1-419 was codon-optimized for eukaryotic expression, synthesized (Genewiz, Inc.), amplified by PCR, and ligated into the EcoR1/Xho1 restriction sites of a modified pPICZ-B vector (Invitrogen). The resulting protein is linked at the C-terminus to EGFP and a 10xHis tag via a short linker (SNS)
followed by a PreScission protease cleavage site (LEVLFQ/GP). Purified full-length protein did not crystallize and was N-glycosylated (data not shown). The construct was modified by PCR to truncate the C-terminal 119 residues and mutate two predicted Nlinked glycosylation sites (N104Q, N108Q) for crystallization. Human TRAAK1-300(N104Q,N108Q)-SNS-LEVLFQ/GP-EGFP-H10 is referred to as TRAAK in the text for
clarity. Vector was linearized with Pme1 and transformed into P. pastoris strain SMD1163 by electroporation. Transformants were selected by plating on YPDS plates with 1mg/mL zeocin. Expression levels of individual clones were compared by FSEC screening of small-scale culture inductions (43). Large-scale expression was performed in a fermentor. Overnight cultures of cells grown in YPD with 1mg/mL zeocin were added to 3L minimal media to an OD600 ~1 and grown overnight at 29°C with glycerol feeding and pH maintained at 5.0 by addition of NH4OH. Cells were then starved to deplete glycerol, temperature was reduced to 27°C, and induction was initiated with slow addition of methanol. Expression continued for ~48-60 hours. Cells were pelleted, frozen in liquid nitrogen, and stored at -80°C"

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