3.5 Large scale BacMam transduction of HEK293 cells in suspension culture

The method described below is particularly suitable for large glycoproteins, which are expressed at low level. For small-to-medium sized and high expression proteins, transducing adherent cells using a few T-175 tissue culture flasks can be sufficient and much more convenient (see Note 15).

Per every 1000ml preparation, 100–200 ml freshly amplified BacMam virus should be used (see Note 16), and here 150ml is used as an example. The virus is ideally within one week from harvest. Pre-warm the virus to room temperature before transduction (see Note 17).

In each 2.8l Fernbach flask, grow 850ml 293 cells to a density of ~1.5×10⁶ cells/ml in Serum supplemented CDM4HEK293 media at 37ºC (see Note 18).

Add 150ml Sema7A or PlexinC1 BacMam virus to the 850ml 293 cells, to make a total volume of 1000ml. Shake at 37ºC for 6–8 hours.

Add sterile sodium butyrate (see Note 19). Continue shaking for 3 days.

Centrifuge the cell culture at 2000g; collect the supernatant, which contains the secreted Sema7A or PlexinC1 protein.