Hi everybody!
I am currently facing a very nasty protein that I am trying to purify. Besides the need for several purification steps, the lack of proper expression in insect cells gives me a hard time actually purifying proper amounts.
So, I started to read up on how to perfectly subculture the cells and also what medium to use. Since I started handling Sf9 cells I have always used Grace's insect cell medium that we supplement with antibiotics, Pluronic (for suspension culture), 10% FBS and L-Glutamine.
Now, I read in several places that serum-free medium (SFM) can yield superior or more protein when using a BEVS in insect cells.
1) Can anyone confirm that? I am unable to find a 100% clear answer if this is usually just the case when you secrete the recombinant protein, if this is true in general or if it just depends from protein to protein which method works best?
2) SFM can be used for secreted as well as cytoplasmic expressed protein as far as I see it. It is not limited to secreted protein expression but advantageous since IMAC can be performed directly?
3) When it comes to the specific BEVS: Is there any system that is superior to the other? Like different versions of DefBac that are used for homologous recombination by transfecting the linearized DefBac together with the rescue plasmid containing the gene of interest (GOI). Or the Bac-to-Bac system that uses the topoisomerase to integrate the GOI into the Bacmid in E. coli that is selectable via blue/white visualization. I also read that stably expressing lines can be established via the so-called InsectSelect system that uses rather early viral promoters and integrates into the host genome. Or again: Does it always depend on your recombinant protein what works best?
4) Is there an easier way to determine virus titer than performing the laborious plaque assay? I need to establish a kind of benchmark or quality control for ideal expression conditions (MOI) for each protein since batches of new viruses may differ in their titer. This control would make protein expression much more reproducible.
I am thankful for every answer, experience and tipps you can give me and looking forward to discussing the topic :)
Cheers