I expressed a membrane protein in pichia. After solubilize the membrane with DDM, I purify the protein by Ni-NTA resin. I run SEC column and found the protein elutes at 9 ml and in the void. This indicates that my protein is possibly in the aggregation form. How should I solve this problem. thanks.
Was the gel filtration equilibrated with DDM? If not, the protein could aggregate when it enters the column and the DDM in the sample is diluted?
If the column was equilibrated wth DDM, was the concentration above the critical micellar concentration (CMC), this could be important for keeping the protein from aggregating or precipitating.
If the DDM concentration was above the CMC, the molecular weight of the micelle (50,000 on average) has to be added to the molecular weight of the protein when considering where the protein will elute. It may be necessary to switch to a resin with a higher molecular weight cutoff, or a detergent with a smaller micelle, such as CHAPS.
If the detergent concentration is not high enough for the concentration of protein, even if the DDM concentration is above the CMC, each micelle may have multiple protein molecules in it, resulting in earlier elution. This is solved by increasing the detergent concentration.
DDM may not be the best detergent for keeping your protein in solution and free from aggregation. You may have to try other detergents.
Gel filtration may not be the best choice of purification method for your protein because of the detergent micelle and aggregation issues. Ion-exchange and affinity chromatography may work out better.
How big is your protein? Considering that many membrane proteins form for example tetramers you might reach a size that runs in the void volumne of S75 columns.
Otherwise Im affraid there is no simple answer. First make sure you are working in a buffer range that is well above or below the pI. Higher salt concentration might help you in case of ionic interaction.
Most likely though is that you will need to screen detergents. Some ionic, non ionic and zwitterionic and see if you get a different SEC profile. Maybe you could do the extraction in DDM and change the detergent on the Ni column.
Good luck
Do you have access to SEC-MALLS? This could be used to determine the state of aggregation.
Hi All, please see my protein on sds-page gel. As you can see, my proteins runs as different oligomeric states including monomer, dimer, trimer, and tetramer. This leads to inhomogeneity of my protein in the solution, so that I may not be able to do the crystallography or EM. my protein molecular weight is about 35 Kda and since it is membrane protein, it runs faster than expected. Could anyone give any suggestions? thanks.
You could use gel filtration chromatography to separate the oligomers, if they are stable. You should run gel filtration in any case to find out whether the oligomer mixture is real or an artifact of SDS-PAGE.
Adam B Shapiro
If the detergent concentration is not high enough for the concentration of protein, even if the DDM concentration is above the CMC, each micelle may have multiple protein molecules in it, resulting in earlier elution. This is solved by increasing the detergent concentration.
I am trying to understand this. My condition is Hepes buffer ph 7.5, 300 mM NaCl, 5% glycerol, 0.015% DDM. Do you think that my DDM concentration is too low, so that proteins aggregate in the DDM micele? If so, I need to increase DDM concentration to 0.03%?
Thanks
What is the range of your SEC column? Don't forget that the DDM micelle has size around 40-70 kDa, so you have to add that to your protein's size.
Is your column equilibrated properly with the detergent?
Hi Hai Li ,
I'm working on membrane protein purification using DDM as a detergent too.
I started the purification of protein with 0.05% DDM in the buffer but I found that the aggregation of my protein was eluted at the void volume in SEC. So, I increased the concentration of DDM to 0.1%, luckily, my protein was eluted at the expected volume but still has some protein aggregation eluted at the void volume. Additionally, I added 1mM DTT to my buffer for preventing the disulfide bond formation during the purification step.
BTW Nawee Jantarit's response reminded me of one thing I did when I was working with transmembrane protein. I would extract the proteins with higher concentration, because there you have plenty of proteins. Then, during the purification, you may decrease the concentration, as the proteins get split into several fractions and diluted.
For that, you may try to extract your protein with various concentrations of DDM (or even other detergents) and see, which condition extracts the most of your protein in relation to total extracted proteins (specific concentration). If you can measure, whether it's active, you should check that as well, to avoid condition which would distort your protein's activity.
Hai Li The CMC is about 0.12 mM (0.061 g/L, 0.0061%)., so 0.015% is about 2.5X the CMC. For a dilute protein, that might be enough to assure one protein molecule/micelle, but for a more concentrated protein it probably would not be enough.
Nawee Jantarit
Yes, thank you for your reply. I was indeed shocked by this. Previously I was purifying membrane proteins, and somehow I get the idea that I should not add too much detergent. I use DDM, and I either do liposome, or nanodiscs. For these 2 years, I have this bad problem that the protein elutes at 10ml, before 11 ml. I use 0.02% DDM in the elution buffer. It really bothers me. I have read many literature, some use 0.02% DDM and some use 0.05% DDM. Then I really that my DDM is too low. So I increase the concentation to 0.05% and the peak starts to elutes at 10.98 ml, and then i increase to 0.06% DDM, the peaks elutes at 11 ml.
Adam B Shapiro , you are right in that indeed I really should use the higher amount of DDM. But I can say that I am really depressed by the early peak. 😂
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