Question about maltose binding protein article 

Hi Victor,

Just now I'm solving this problem with respect to this maltose binding protein tag, I found this article on wiki. And here is what you wrote:

"...Once the fusion protein is obtained in purified form the protein of interest (X) is often cleaved from MBP with a specific protease. Protein X can then be separated from MBP by passing through the amylose column again."

It would be great if you can explain how can "Protein X can then be separated from MBP by passing through the amylose column again" because I have tried it and it doesn't work. My personal guess is since MBP binds to maltose, it simply doesn't bind to amylose resins / column unless maltose is removed beforehand.

Then the question is how to remove maltose from MBP. From Davidson et al. Mechanism of maltose transport in Escherichia coli: Transmembrane signaling by periplasmic binding proteins (1992:

"Preparation of Maltose-Free MBP

MBP was purified by affinity chromatography and was dialyzed extensively under conditions calculated to remove most of the bound maltose. For some experiments, MBP was denatured in 6M guanidine hydrochloride (Gdn-HCl) and either dialyzed against 6M Gdn-HCl or passed through a Sephadex G25 column equilibrated with 6M Gdn-HCl before renaturation by dialysis against 10mM KP1 (pH 7.0). UV-difference spectroscopy showed that the bulk of the MBP was free of maltose."

Hope to hear from you soon.

Martin