MBP tagged protein not bind resin - (Jul/27/2005 )
does anyone use MBP tag the protein to make it soluble. now my problem is the protein is soluble. but seems not bind to the column. really make me desperately
any suggestion will be appreciated
I've experienced fairly weak binding of MBP to amylose resin. One of the major problems we've noticed in our lab is that once the resin is used once, it's tough to get anything to bind again. It seems that the maltose binds tightly to the resin and prevents binding of MBP on subsequent runs.
Check this website for a regeneration protocol...
The section is about halfway down the page, under "regeneration of amylose resin".
If you're having this problem with fresh beads, you may want to try a batch binding step - put the resin in a tube with your protein and nutate for several hours at 4 degrees C. Then either transfer to a column or spin the beads down and add maltose to remove the protein.
Edit: I had my facts mixed up on this one - it's not maltose binding to the resin, it's maltose binding to MBP that's tight. Trying to remove MBP after it's been exposed to maltose tends to be a large problem because the binding site gets blocked, no matter how much dialysis you do.
thank you so much.
Actrully I was bind the resin overnight 4 degree, then load to the column. I got my band once when induction 30 degree, but with another extra band. so i tried different temp to induction, same procedure for purification, i run the SDSPAGE can see the protein is induced, but after run through the column, nothing is there. really annoying me. do you have any idea what the problem might be?
When you say "nothing is there", what exactly do you mean? Is it nowhere at all? Have you checked your flowthrough, wash, and all eluent fractions? Your protein obviously won't just disappear, so it's got to show up somewhere. If you don't see it in any of the partitions, it may be binding too tightly to the column, and you're just not getting it off (try increasing maltose concentration in elution buffer). If it's coming off in the flowthru, it's just not binding to the column, in which case you may have contaminating maltose on the resin (try the regeneration protocol).
Another thought - assuming this is your first purification step, and you're loading soluble extract directly onto beads overnight, the difference in growth temperature could affect the amount or activity of proteases in the solution, which could chew up your protein given a long enough incubation. You could solve this by using more protease inhibitors (PMSF, EDTA, maybe a "complete protease inhibitor tab") during lysis.
Thank you so much for anwsering me. It is really helpful. do you think add some DTT in to lysis buffer would increase the chance to bind teh resin?
by the way, I am doing a zinc finger protein, could i add DTT into it. I know DTT break cys-cys bond, is it going to have effect on zinc finger protein?
Not directly, but DTT does chelate metals, so you'd lose the zinc. If you're just looking for a reducing agent, beta-mercaptoethanol shouldn't interrupt a cys-Zn bond. Unless your protein is unstable without the Zn, though, you can probably add it back in later.
I have no reason to believe that the presence of reducing agents is going to improve your fusion protein binding to the column, though. The column works by impersonating maltose, not through anything sensitive to oxidation. Do you have any particular reason to believe DTT would enhance binding?
Have you checked for the presence of your protein in all fractions yet? Is it a degradation problem, or simply a non-sticking problem?
I would be concerned that your protein is being digested by proteases during your o/n binding. Do you get protein with shorter incubation times?
DNA sequencing software
i do not think it was digested, i seee it in the flow through. One of the protein i did the same way and it is ok. but this one seems trouble binding.
Well, if you see it in the flowthrough, the most likely explanation is simply that your MBP fusion is being contaminated with maltose somewhere along the way. I'd use fresh resin, scrupulously clean glassware (scrub it out yourself and autoclave it, or use new materials), and new buffers.
If you've done all that already, I'm out of ideas, aside from the possibility that you're getting only partial proteolysis of the maltose binding site. In either case, if the "all fresh" method doesn't work, I'd just use your original condition (30 C induction) and add in additional chromatography steps to try to remove your extra band (there's a strong possibility it's just extra MBP that ended translation before it got to your coding frame - ion exchange and gel filtration may be sufficient to remove it).
Or, failing that, try engineering a his-tag somewhere on the protein as well - either at a terminus or inbetween the fusion and the protein, then purify via Ni-affinity.