Hi all, with MBP tag at N-terminus and 6xHis at C-terminus, I have been trying 5 protein expressions in NEB Exp cells, optimized at different temperatures and IPTG conc. Upon Ni-NTA purification, I'm seeing several truncated bands with strong MBP signal and very weak band at right size bands with several intermediary sizes on SDS-PAGE. I avoided to use amylose resin because mpb is at N, and only using Ni-NTA (to avoid pulling down non-full length molecules.
I used pMAL-c5x several times with several proteins (from 10 kD to 350 kD proteins) in the past with very good success, but never saw this happen. If anyone faced this problem and solved it, your insights would be highly appreciated. Thanks!
This can happen for specific proteins. There's something about the structure that E. coli really doesn't like. We'll never know why. But I can assure you it's statistically about time that you run into one of these difficult ones. Can you live with the amount of full length protein you're getting?
If you do a sequential purification with Ni first, amylose second, you should be getting only full length proteins. Otherwise you would have to have some very weird internal deletions going on, like intein splicing ?! Are you sure your Ni washes are stringent enough? With 40 mM imidazole in bind & wash buffers, or better gradient elution?
Does the pattern suggest that there is cleavage between MBP and your protein? If so, I would check the sequences (both vector-derived and insert derived) that link your protein to MBP. Maybe there is something that you can eliminate at the junction. this sometimes helps. for example: there is a long stretch of Gln and Ser residues and a TEV cleavage site. Should you not plan to use TEV protease, these residues can be eliminated.
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