Dear Expert Science community,
I am interested to express a bacterial protein on the mammalian cell surface. As in mammalian cells, proteins are sorted by ER and golgi compartment. How should I proceed?
Waiting for your kind suggestions!!
Thanks
Should be feasible and work with a little luck, Sandeep.
Adapt codon usage to mammalian (otherwise, expression levels will be very low), make sure there is a Kozak Consensus Element for starting transcription at the right place and appropriate signal sequences, a transmembrane domain (orientation!) and possibly a spacer that makes all of your protein accessible. pSecTag2A might be a useful plasmid for your project, as it brings most of the elements you'll need, but with affordable gene synthesis nowadays, it's not that critical.
Be aware of accidental unwanted posttranslational modifications (e.g. glycosylation), and there might be some risk of misfolding, so you'll maybe have not the desired activity.
When you have done the in silico stuff, have your cDNA synthesized and subclone it into a mammalian expression vector.
Mammalian Expression Systems. Bacteria have no nucleus, endoplasmic reticulum, or golgi apparatus, which are all key elements in cellular transport and post translational modification. ... In contrast, eukaryotes like mammalian cells possess these organelles and the molecular machinery that comes with them.High-level stable and non-replicative transient expression can be carried out in most mammalian cells. The vectors contain the following elements: Human enhanced cytomegalovirus immediate-early (CMV) promoter for high-level expression in a wide range of mammalian cells.
I think you can. It is a new technique and not very much used, it has many limitations. I wish I could support my answer with a paper. I think it is expensive and not all proteins are allowed.
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