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!