Hello All, I would second the notion that transient systems are quite good for testing expression levels on the small scale and can be used for scale up (to a certain degree at least). It is probably the fastest way to go to screen a number of constructs.
Cheers, tom -----Original Message----- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of r...@mrc-lmb.cam.ac.uk Sent: Sunday, 22 October 2017 10:27 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Large scale mammalian expression system recommendation? Hi Nate, There can be no consensus I guess, most importantly because transmembrane proteins are so diverse :-) We typically use transient transfection at the screening stage, and often for large-scale expression (e.g. PMID 24909990). Bac-Mam has some advantages (nicely described in 25299155, 27041595), but it's obviously more time consuming. Lentiviral systems (we use Clontech) are also great. But again the target often determines the choice of system. Whether it contains multiple subunits, whether overexpression has a negative impact on cell health (a tetO switch can be helpful, 12370422). Also, I'd use different systems depending whether the recombinant protein is intended for functional, fluorescence microscopy, structural analyses (say X-ray vs cryo-EM)..... Best wishes, radu -- Radu Aricescu MRC Laboratory of Molecular Biology Francis Crick Avenue Cambridge Biomedical Campus Cambridge CB2 0QH, U.K. tel: +44-(0)1223-267049 fax: +44-(0)1223-268305 www: http://www2.mrc-lmb.cam.ac.uk/group-leaders/a-to-g/radu-aricescu > Hello everyone, > > I wonder if there is a consensus for what is currently the best system to > express transmembrane proteins in mammalian cells? > > I think that baculovirus transduction ("Bacmam" anf the likes) has been > used historically more (?) but would like to know if the modern adenovirus > systems offer any advantages in terms of expression levels. I used both in > the past but not for transmembrane proteins and never compared them back to > back for the same protein. > > Has anyone attempted a direct comparison? (It has to be mammalian cells, so > baculovirus/insect cells won't do). > > Thanks for any comments/insights, > > Nate >
