pPICZ-C-coreTAPL

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	Transformation of the protease-deficient His+ P. pastoris strain SMD1163 was performed by electroporation as described (1, 2) (1.5 kV, 25 μF, 400 Ω, Gene Pulser II; Bio-Rad), using 50 75 μg linearized DNA. Transformants with highest expression of both coreTAPL constructs were selected as described (1, 3, 4).

Protocol tips
Transformation of the protease-deficient His+ P. pastoris strain SMD1163 was performed by electroporation as described (1, 2) (1.5 kV, 25 μF, 400 Ω, Gene Pulser II; Bio-Rad), using 50 75 μg linearized DNA. Transformants with highest expression of both coreTAPL constructs were selected as described (1, 3, 4).

Publication protocol

A single-cysteine variant of human coreTAPL (Q9NP78 UniProt, amino acids 143–766) and a coreTAPL fusion construct were designed for expression in Pichia pastoris. Both constructs were flanked by EcoRI/XhoI restriction sites for cloning into the pPICZ-C vector (Invitrogen). The fluorescent version of coreTAPL (coreTAPLmVenus) was C-terminally connected by a linker (STNLGSENLYFQGVAIGGLAV) with the YFP derivative mVenus followed by a second linker (GLDAAGGGGSGGGGSLV) and a C-terminal His10- tag. The single-cysteine mutant of coreTAPL, which shows the same activity as wt TAPL, is connected by a single serine with the C-terminal His10. All four cysteines of coreTAPL were substituted by alanine or valine, and a single cysteine was introduced at position two. Transformation of the protease-deficient His+ P. pastoris strain SMD1163 was performed by electroporation as described (1, 2) (1.5 kV, 25 μF, 400 Ω, Gene Pulser II; Bio-Rad), using 50 75 μg linearized DNA. Transformants with highest expression of both coreTAPL constructs were selected as described (1, 3, 4). Largescale expression in a 7.5-L Labfors4 reactor (Infors HT) was performed by fed batch method (3). Cells were frozen in liquid nitrogen and stored at −80 °C.

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