Dear all,
It have been reported repeatedly by many labs to make crystallisation of target proteins possible only by fusion of maltose-binding protein (MBP) to the N-terminus of their targets. We have tried such strategy on a large number of our proteins (>30), but none of them crystallised, despite that all fusion proteins behaved very well upon purification (high solubility/homogeneity).
Any of you have successful stories to share in this regard? FYI, we mutated a few bulky residues in the last helix of MBP to Ala as reported by others, and used a linker of two or five Ala between MBP and our targets. Additionally, maltose was always supplemented to the purified proteins before setting up droplets.
Thank you!
G. Dong
Gang,
Yes, a MBP tagged construct was critical for the crystallization of MCL1 in the desired context.
Best,
Stefan
Article A Maltose-Binding Protein Fusion Construct Yields a Robust C...
Go to www.rcsb.org advanced search and combine a text search for "Maltose Binding Protein" with a text search for "Fusion", using the "And" logical operator
In the long run, it is less work to tell people how to find the solution to a problem, than to give them the answer directly.
Yes Annemarie, but how do you know they are in it for the long run?
If you always wait until you are absolutely sure, you'll never get anything done.
MBP is frequently added to recombinant constructs to improve soluble expression and as an affinity tag for purification on an amylose column. So if you anyhow use an MBP fusion to facilitate production of your protein, you might as well try the fusion in crystallisation.
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