I am finding it difficult in a large scale purification to use this tag. Also, I am not getting optimum expression of my protein by inducing at both 37 and 18 degrees as well. Rosetta DE3, C43 and C41 cells have been used. Could someone suggest a better protocol for expression and purification!
It wouldn't necessarily be my first choice, since you wouldn't end up with "native" protein, but you can use substrate analogs to label serine proteases with a pull-down tag. It would be highly effective and specific, but at best you're still going to have the active site covalently linked to a strange molecule.
On the other hand, you are guaranteed to only be extracting protein in an active conformation, so you will be excluding aggregates, misfolds, etc, so that is attractive if the active site isn't your primary target of interest.
As long as the serine proteinase doesn't cleave his tags, these might be best. The mbp and GST tags are proteins that will be cleaved possibly by the serine proteinases. Other tags are HA, FLAG, and you can check the sequences to make sure they aren't cleaved. Then there is biotin, but it is hard to elute to get purified protein, but there are ways around this.
In terms of expression, I have no idea. It is usually empirically determined for a specific protein as to what is best.
- Badges
- Science topic
- Similar topics
- Chemistry
- General Biochemistry
- Proteomics