Protease in expression vector - (Jun/16/2005 )
When those expression vectors come with the protease (Xa, thrombin etc), is the tag cleavage constitutive, I mean, does the tag cleavage happen by itself? If I want to retain the tag (for elution purposes, detection etc), can I use a vector that has the protease or not?
Probably I need to update this information since it is my first time to hear that a protease presents in the vector itself for cleavage purpose.
As I understand, the tag is require for purification purpose, if the cleavage happens just during expression (due to the co-present of protease and the cleavage site in the vector) then I wonder what is the use of the tag?
Or I think I misunderstood your message!
Your question seems to be a little strange. Generally the vectors come with cleavage points for protein tags. Quite often the tags are retained as they do not interfere with the various studies. However, undercircumstacnes where its prresence is unwelcomed, it can be removed by treatment with the speified protease. This treatment is generally done after purifcation.
as it was said before, vector which carry this kind of sites for protease cleavages, are so to allow the cleavage after purification of your protein by affinity to the tag they are expressed with.
If your question is because you are seeing your protein without the tag, you should check for the bacteria you are using.
Usually the vector company manual propose the appropiate strain to express your protein.
IF there is a protease cutting the tag when you are expressing, is not from the vector, it is endogenous from the bacteria.
So check this!
Then if you ask this because you cannot purify your protein, maybe it is a folding problem, where the tag becomes not exposed to couple to the column.