Protocol

Abbreviation
WU_PEI_transient_transfection
Name
WU_PEI_transient_transfection
Laboratory name
Washington University
Type
expression
Description
Large-scale Transient Transfection of Mammalian Cells in Suspension Culture. The majority of targets produced by the CSGID are expressed in E. coli as soluble-natively-folded proteins. This works well for most prokaryotic proteins and for many eukaryotic proteins that normally reside in the cytoplasm and that can fold properly in the absence of post-translational modifications such as carbohydrate addition, disulfide bond formation, or enzymatic cleavage.

To increase the range of protein targets available for structural studies we have been exploring high-throughput protein expression in mammalian cells by transient transfection. The system has several properties that make high-throughput production of protein achievable. 1.) Large numbers of cells can be transfected using high-density suspension culture. 2.) Rapid recovery of purified protein from serum-free and protein-free media cultures. 3.) High transfection efficiencies can be achieved using an inexpensive cationic polymer polyethylenimine (PEI). 4.) Peak protein production occurs within 3 to 6 days of transfection. 5.) It is also possible to SeMet label proteins by this method.

Additionally, alternative versions of this protocol have been developed to minimize the extent of linked carbohydrate. These involve either protein expression in medium containing glycosylation inhibitors (like kifunensine), or protein expression in cells that lack the ability to make complex N-glycan (like HEK293-S cells, ATCC CRL-3022). For both methods the remaining carbohydrate is trimmed by digestion with Endo H.

Cell preparation for protein production. The principal cell line we use for protein production is Freestyle™ 293-F, a sub line of HEK293 that has been adapted to suspension culture (HEK293 cells are a permanent line established from a primary human embryonic kidney cell culture by transformation with sheared human adenovirus type 5 DNA). We obtain 293-F cells from Invitrogen and maintain them in serum-free protein-free medium (GIBCO® FreeStyle™ 293 Expression Medium, Invitrogen Cat # 12338) supplemented with Penicillin-Streptomycin (Invitrogen Cat# 15140). Typically these cells grow to densities of 1-3 x 106 cells/ml and need to be split every 2-3 days. For transfection, we grow 200 mls of cells in a 1L vented-cap erlenmeyer flask using an orbital shaker housed in an 8% CO2 incubator at 37 °C.

Expression construct design. Proteins targeted to the ER, Golgi, or secretory pathway must be inserted in an expression construct in frame with an N-terminal signal peptide. A signal peptide is unnecessary for proteins to be expressed in the cytoplasm. In either case, the starting MET codon must fit the Kozak consensus sequence. We have had good success with C-terminal 6His, human IgG2-Fc or mouse IgG2b-Fc tags. Protein purification is often followed by TEV cleavage to remove fusion tags. Nearly any mammalian expression vector containing the human cytomegalovirus (CMV) promoter regulatory region may be used to drive target protein expression in 293-F cells.

Reagents. The PEI (Polysciences Cat# 23966) is prepared for use by dissolving 50 mg in 50 mL ddH20 and then adjusting the pH to 7 with HCl. After filter sterilization, the PEI is stored at -20°C. Standard Qiagen DNA preparations are suitable for transfection.

Transfection protocol for suspension cells.
In brief,
1) Before transfection, cells should be ~1 x 106 cells per ml (200 ml/1L shaker flask).
2) Mix 200 ug DNA with 5 ml serum-free antibiotic-free medium (we use Invitrogen Opti-MEM Cat# 31985) and vortex briefly to mix.
3) In a second tube mix 300 ug PEI solution (1mg/ml) with 5 ml serum-free antibiotic-free medium and vortex briefly to mix. (The optimum DNA:PEI ratio varies for each construct, but is usually around 1:1.5 or 1:2)
4) Mix the PEI and DNA solutions together and wait for 10 min at room temperature to allow the DNA-PEI complexes to form.
5) Add the transfection mixture to the cells and incubate at 37°C for 3 days before harvesting. For secreted proteins, replace the supernatant with 200 mls of fresh medium and continue the incubation for another 3 days.
6) For cytoplasmic targets, lyse the cell pellet with detergent and purify the protein. For secreted targets, pool the cell supernatants and purify the protein.