#CAP-T cells are emerging as an alternative to #CHO and #HEK cells for protein expression considering some inherent genetic aberrations in the latter ones, hindering expression of certain classes of proteins. Dive in for an introduction to these amazing cells!
#CAP-T cells are a modification of #CEVEC's Aminocyte Production (CAP) cell line by transfecting them with E1 and pIX functions of #Adenovirus 5. They also constitutively expresses #SV40 large T antigen causing transgene expression by #episomal replication and maintenance of expression #plasmids carrying SV40 origin of replication. This is advantageous as it allows working both at early development stages (using TGE on CAP-T cells) and production levels (using SGE on CAP cells) without the need of changing the cell line.
Why CAP-T cells?
In different #CHO sublines, genotypic observations lead to disability of protein expression by loss of #chaperones or other processing enzymes.
Also the genetic repertoire of #HEK293 cells/ kidney cells could be inherently incapable of supporting expression and processing of certain #genes of different #tissue origin.
Transient Expression protocol:
Day -1: Grow #CAP-T cells on Protein Expression Media + 4mM #GlutaMaxI (PEM+G) media at 37C, 5% CO2 and 120 RPM (orbital shaking diameter 25mm).
Day 0: 1. Take Cell count, calculate the volume for 20 million cells, centrifuge the culture at 200g for 5 mins and re-suspend the cells in 4mL of #Freestyle293 media supplemented with 0.1% #Pluronic F68.
3. Dilute #PEI Max reagent as 3pg/ cell similarly, vortex 10s.
4. Add #pDNA solution directly to the CAP-T cells, incubate 5 mins at RT.
5. Add diluted #PEI Max solution to the culture, incubate at 37C for 4h.
6. Add 24mL PEM+G and 4mM #valproic acid (to enhance protein expression by inhibition of histone deacetylase) to the culture after 4h incubation.
Day 3: Take regular cell count and viability check. Add feed to the culture as per the following table:
By Day 10 culture viability is expected to drop below 80% and that is the time you harvest your culture.
Harvest Day: 1. Spin the culture at 4000-6000 RPM for 30 mins at 4C.
2. For secretory protein, harvest the supernatant and add 0.02-0.03% sodium azide and Protease inhibitor, filter sterilize and proceed to purification.
3. For intracellular protein, wash the pellet with #PBS and proceed to purification.
More to munch on: