Hi everyone!
I currently have the issue that my proteins are all eluting at the salt wash that I apply during my IMAC (HisTrap 5ml HP column).
My proteins are all His-SUMO-tagged. They are followed by a highly charged domain that should have a structure (alpha-helical but with a small unstructured region as well) and is very positively charged. After that, there is a domain that binds the cell wall of bacteria and another domain that should be cleaving the cell wall of bacteria.
I have been working on this type of protein for the last two months, and the only thing that is different among all constructs is just the positively charged domain between the N-term His-SUMO-tag and the other two domains on the C-term.
Usually, I load the sample in 20mM HEPES pH 7.4, 300mM NaCl, 1mM DTT, 5% Glycerol, 20mM Imidazole on the column, perform a wash with the same buffer with 50mM Imidazole and then another wash with 1M NaCl containing 20mM Imidazole. My first constructs were totally fine with this procedure and did not elute in the salt wash (just in the actual elution with 300mM Imidazole).
Then, after I tested different constructs that differed in the charged domain, I started seeing that every construct I test, is eluting in the salt wash. The only thing that changed, besides that one domain, between those constructs is that I started expressing the latter ones in TB autoinduction (25°C, 1.5% Lactose) due to time reasons (just too much constructs to express and test). Before, where I saw no issues, I was expressing in LB at 16°C with 0.1mM IPTG.
Might this be one reason that these constructs now behave so strange? The expression in TB always looks perfect. A lot of protein. But might this be the issue? Is it too much protein? Are they aggregating already? Does this change anything concerning electrostatic interactions? Does anyone have experience with this issue?
I have to mention that we started implementing a high salt wash in every E. coli purification since we always have a high 260nm signal. So, my idea would be to simply perform a normal IMAC without a salt wash in the future. Then, to remove the DNA in the flow through, I could perform a cation exchange (constructs are all around pI 10). Or would you rather actively bind the DNA with an anion exchange and collect the protein of interest in the flow through? Then, I cleave the protein with SUMO protease and remove the tag via size exclusion.
Alternatively, I heard that PEI could be used to initially precipitate in the lysate DNA? I just know this reagent from using it as a transfection reagent.
Benzonase is in our lysis buffer anyway, but it doesn't seem to help the removal of nucleotides in our sample.
Again, I am happy for insights, suggestions and fruitful discussions.
All the best!
"perform a wash with the same buffer with 50mM Imidazole and then another wash with 1M NaCl containing 20mM Imidazole." I guess 50 mM imidazole in wash buffer may not be appropriate for wash (i.e., imidazole concentration is too high), and would suggest using < 30 mM imidazole concentration for wash. Bedsides, I would also suggest using 5 mM beta-ME instead of 1 mM DTT in your buffers. Hope this will be helpful. Thanks.
Actually, we do wash with just 20mM Imidazole prior to the salt wash and this does not help. I wrote down the 50mM Imidazole wash before the salt wash here in this question since this is what we will do in the future since these proteins are quite sticky anyway. Therefore, 50mM Imidazole wash is totally fine and more than enough protein is coming down in the elution if there is no salt wash. However, with the salt wash, if elutes there already. What is the exact reasoning for Beta-ME? Why this and not DTT? Or even TCEP? Any particular advantages?
All the best and thanks for the answer!
If the wall binding domain has a positive charge and the Histag has a negative charge (at pH 7.4), they may bind to one another. Raising NaCl content (0.5M? 1M?) from the loading step may reduce electrostatic interactions and allow the Histag to bind the column.
You can also increase the imidazole gradient up to 0.5M, just in case the Histag gives you more trouble.
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