I have a MBP fusion protein that is not binding to amylose beads. I have already tried adding glucose to the media (although I did not add it to my starter culture) and I am not using detergents or salts in my buffer. Has anyone found methods to increase binding to the beads? I am wondering if my protein is forming some large complex with nucleic acids (it is known to bind nucleic acids) and other proteins and the MBP tag is obscured.
I agree that it's a possibility that your protein may be in complex with DNA and other proteins that may mask the MBP tag. If that's the case, treatment of your sample with DNases may help. Also if I remember correctly, hydrophobic interactions are involved in MBP binding to maltose. So, salt may enhance its binding affinity and specificity to its subtract, and thus adding salt to your binding buffer may also help.
From my reading the binding occurs through hydrogen bond interactions as well. I will consider the use of DNases, thanks.
I have always added a His tag before MBP in all of my MBP fusion proteins.
This helps a great deal in purification and also getting rid of MBP once you cleave the tag.
Hi! I would suggest you to do first a IMAC purification using His and then purify your fusion protein using amylose resin =). In our lab we never got binding to amylose beads without a first step of purification. Good luck
Thank you for your suggestions! I can't use a His purification or salt unfortunately but I think using benzonase helped!
As an update, I was able to get the His6-MBP tagged fusion protein to bind to amylose beads. I had to implement a purification step prior to binding to amylose resin, although I did not have to use IMAC as the purification step.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Nucleic Acids