I have 2 proteins cloned in PET 41a which contains GST tag. To cleave the GST tag I need to digest the expressed recombinant protein with enterokinase. I tried 2-3 times with proper calculated concentration of enzymes but unable to cut the GST tag. Is there any method to cleave the GST Tag from the protein cloned in PET41a vector. Enterokinase is quite costly so I am looking for an alternative way to remove the GST Tag from my protein. Can anyone suggest anything about this problem?
Have you made sure that the enzyme site is intact after your cloning?
Put a TEV protease site. you can purify your own TEV protease quite easily
Yes definitely Entrokinase is active.I have calculated the amount of enzyme and put reaction but unable to cut the site..
- Are there reducing agents in the buffer? They inactivate EK.
- Is a proline or tryptophan residue at the P1' position? Those are not good for EK.
- Perhaps cleavage is sterically hindered. Try cutting in 1 M urea.
@ Alejandro : if i cut the entrokinase site in presence of 1 M urea, there is a possibility to degrade the protein as well...
You mean, by mechanisms other than the activity of enterokinase itself? Can you clarify a bit?
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