pIEX-5-6His-mMBP-UMODpXR

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	Transfection mixes were prepared by first combining serum-free medium and DNA, then adding PEI, and finally incubating the resulting solution for 10 min at 20 °C before adding it to the cells.

Protocol tips
Transfection mixes were prepared by first combining serum-free medium and DNA, then adding PEI, and finally incubating the resulting solution for 10 min at 20 °C before adding it to the cells.

Publication protocol

"DNA for transfection was prepared using HiSpeed Plasmid Midi/Maxi kits or EndoFree Plasmid Giga kits (QIAGEN). Human embryonic kidney (HEK) 293T or 293S (ATCC CRL-3022) cells were amplified, transferred to a suitable expression vessel and transiently transfected using 25 kDa branched PEI, based on a published protocol (Aricescu et al., 2006). Fresh DMEM medium supplemented with 4 mM L-Gln (Thermo Fisher Scientific) and – if required – 2% fetal bovine serum (Biological Industries) was added to 90–95% confluent cells using the volumes specified in Table S1 for transfection of 6-well plates (10 cm2/well; Corning), T-flasks (150 cm2; BD Biosciences), ribbed roller bottles (2125 cm2; Greiner) or cell factories (2528 cm2; Thermo Fisher Scientific). Transfection mixes were prepared by first combining serum-free medium and DNA, then adding PEI, and finally incubating the resulting solution for 10 min at 20 °C before adding it to the cells. Unlike HEK293T cells, HEK293S cells tend to detach from ribbed roller bottles and were thus preferentially grown in T-flasks or cell factories. Medium was harvested three days after transfection.

For intracellular protein production, 200 ml FreeStyle 293-F HEK cells (HEK293F; Thermo Fisher Scientific) were grown in suspension in FreeStyle 293 Expression Medium (Thermo Fisher Scientific) in a 1.0 L plastic shake flask (Corning), using a shaking 8% CO2 incubator. When cells reached a density of 2.5–3.0 × 106 cells/ml, they were transiently transfected by diluting them 1:1 with medium containing 3.0 μg/ml plasmid DNA and 9.0 μg/ml 25 kDa branched PEI. Harvesting was performed four days after transfection."

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Manufacturer protocol

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