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Tehn 02-19-2012 11:49 PM

Advice on soluble aggregate problem
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!

eeuzc 03-03-2012 11:49 AM

Re: Advice on soluble aggregate problem
From what I understand, you are having problems trying to separate your protein of interest from the tag. You could use affinity chromatography matrix to do this so that your tag and any remaining tagged protein is removed to give you untagged protein.

Although I would suggest you add more TEV or increase digestion time to increase your protein yield. Also, is there a problem in the protein solubility once the tag is removed? You could use higher salt concentration in your buffer to solubilize your protein of interest.

Hope this helps!

fit2page 07-28-2012 05:02 AM

Re: Advice on soluble aggregate problem
veru useful information and tips

Kneeanderthal 10-18-2012 09:26 PM

Re: Advice on soluble aggregate problem
I agree with the first comment about increasing digestion time. Have you tried cleaving at different temperatures? This might help improve cleavage efficiency. Try 25 C (overnight) in cleavage buffer and 4 C (two days) in cleavage buffer. what is the percentage of recombinant protein in the lysate? If is pure enough you could just cleave the protein before placing it on the column and remove the tag by passing it through the column.

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