Could anyone help me to define a range (in mg) of expressed protein in mammalian cell after transfection. We use a plasmid with CMV promoter and would like to know what is the values consider as high, good or poor protein expression.
Any ideas are welcome
Thanks
Our in-house plasmids also have a CMV promoter.
If your protein is a very resistant one, typically an antibody, you can expect at the very least tens of milligrams per litre when working with easy-to-transfect cells like CHO, HEK293, and derivatives. It is evidently dependant on how far you have gone in optimising your culture and transfection protocols (feeding strategy, culture medium at transfection, transfection vector, downstream processing and purification, etc.). Best case scenario, a transient transfection can yield over half a gram per litre.
Even though, for demanding proteins (e.g. cytostatic ones) a few milligrams per litre could turn out to be a success.
Through my occasional use of this technique, I have experienced yields ranging from 0.5 mg/mL to 50-80 mg/mL, with a typical 20-30 mg/mL through hardly any further optimisation from the protocol that I started with. (no mAbs nor cytostatic proteins here)
http://link.springer.com/article/10.1385/MB:34:2:225
hi,
I only wonder if it is possible to define a range ( max or average) of expression for this kind of system?
I agree with both Paul and Alexander. Actually protein yield also depends on the cell line and the transfection efficiency etc.
Hi they are right it will be highly dependent on your expression cassette (plasmid), the protein you want to express, cells, culture conditions such as volume of culture (bioreactor) etc.
Thanks everyone, could you share any references for this kind of experience?
Our in-house plasmids also have a CMV promoter.
If your protein is a very resistant one, typically an antibody, you can expect at the very least tens of milligrams per litre when working with easy-to-transfect cells like CHO, HEK293, and derivatives. It is evidently dependant on how far you have gone in optimising your culture and transfection protocols (feeding strategy, culture medium at transfection, transfection vector, downstream processing and purification, etc.). Best case scenario, a transient transfection can yield over half a gram per litre.
Even though, for demanding proteins (e.g. cytostatic ones) a few milligrams per litre could turn out to be a success.
Through my occasional use of this technique, I have experienced yields ranging from 0.5 mg/mL to 50-80 mg/mL, with a typical 20-30 mg/mL through hardly any further optimisation from the protocol that I started with. (no mAbs nor cytostatic proteins here)
http://link.springer.com/article/10.1385/MB:34:2:225
Transient transfection of CHO and HEK293 lines and their derivatives is becoming increasingly popular in biopharma industry as a way to rapidly produce large batches of therapeutic proteins (primarily secreted antibodies and growth factors) for pilot R&D studies. The best reported yields are in the 1-2 g/l range, a few 100s mg/l are fairly standard, however those levels can be only achieved after extensive optimization. There are also some commercial CROs offering such service, for example Icosagen.
The average of protein yield is depends on the copy of genes expressed by the host system. Even the respective genes are under the control of CMV which is a good promoter for the expression of genes in mammaliancells we can not predict the amount of protein. There are avanced strategies were came to increase the expression of genes. Please go through it.
all the best
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology
- Culture Cells
- Cell Line