Advice on soluble aggregate problem - (Feb/19/2012 )

Hi all,

I'm having an interesting and annoying problem with my latest protein of interest. It is ~10kDa in size and so far I have only been able to get soluble expression when it is fused to HisMBP, a 40kDa affinity/solubility tag. I remove this N-terminal tag by cleaving with TEV but this process does not go to completion. I end up with three species, the full length fusion protein (HisMBP-Prot), the cleaved tag (HisMBP) and the cleaved protein itself (Prot). Running this mixture on any of the size exclusion columns my lab has access to gives me two peaks, one containing the cleaved HisMBP tag at the expected size of 40kDa and another in the void volume which contains HisMBP-Prot and Prot.

This soluble species cannot be removed from solution by spinning at 16000xg or by a 0.22 um filter. I have tried refolding this species but it apparently reforms at the same size.

Right now I'm thinking about the next steps to try and get this thing in a monomeric form and any advice would be much appreciated, even untested ideas. I am planning to vary the pH (currently 7.3) and up the salt concentration (current buffer is 100mM NaCl, 25mM NaPhos, 5mM B-mercaptoethanol) to try to break it up.

Thanks in advance for any help!

Hi,

I've dealt with the same issue (http://www.protocol-online.org/forums/topic/22454-removing-mbp-tag/page__p__117064#entry117064) and, I'm still working on it. Besides things I mentioned in the forum I tried to break the aggregates with Acetonitrile (tried 10 - 40 %) and used PEG 6000 which should help preventing aggregates. Furthermore I tried to "elute" the cut protein from the uncut by low pH-buffer (~4) and high pH-buffer (~9)
For my protein nothing worked out yet but maybe it will help you. I also noticed that the extent of the aggregates differ with every protein purification (checked it by nondenaturing gradient gel).

Wish you good luck!

Hi,

Thanks, it's good to know that I'm not the only one having this issue. I will definitely give the PEG and acetonitrile steps a try. I have managed to separate the cut and uncut protein by denaturing them in 6M Guanidinium Chloride and pulling out uncut with nickel but after refolding the cut protein I have very little left and I'm not sure if it's in the correct form. I will let you know if I come across anything useful.

Good luck to you too!