I am trying to purify an endonuclease (N-terminal hexahistidine tagged) of about 16kDa. The first round of purification involves overnight binding with Ni-NTA beads. When the elution fractions are pooled and injected for size exclusion chromatography, there is always a problem.
1. Initially, my protein used to elute out in the void, that could possibly suggest aggregation, or an oligomeric state of high molecular weight. The yield was also very low.
2. I had eventually begun adding NaCl to the buffers during batch purification.This slightly improved the yield, but after a gel filtration round, I see no protein at all. (Tried with both Superose 12 and Superdex 200 columns).
This has been totally baffling me as the columns are working perfectly fine and yet I can't find the reason for my protein loss. My questions are:
1. What could be the reason for this loss of protein? I have read somewhere that sometimes the protein aggregates too much and non-specific adsorption may take place, resulting in protein loss. How do I eliminate that possibility? Can anybody tell me more about this problem?
2. And, if size exclusion is to be completely eliminated for purifying the protein, then how else can I purify the protein? Ion exchange is not working either. Is there anything else I can try?
Thank you so much!
It could be useful to know steps, buffer and timing of the purification.
10% glycerol in all buffers might help. A different pH might help, however, nickel chromatography requires a basic pH.
It is a DNA binding protein, therefore aggregation might be caused by binding of the protein to DNA (or RNA). Add a nuclease to the lysis buffer.
Especially small proteins are often more soluble when fused to a larger tag such as MBP.
Hi Sara,
as others have said, you need to provide more information to receive more help. Anyway, the recommendations from Konrad could work.
When you attempt to purify your protein, you need to pay attention to:
- features of the protein: size or MW, charges (isoelectric point or net charge at specific pH values), existence of specific ligands (that can be used in affinity chromatography)...
- features of the major contaminants (what from the above differentiates them from your target protein?)
- features of the medium: concentration of your prot and of other contaminants, salts, etc.
Choose your purification procedures based on this information. If you dont know it, study your sample first using SDS-PAGE and IEF in you can. A titration curve also gives a lot of information.
You mention that Ion Exchange didn't work. What did you try? IEX is too broad (cation, anion, strong, weak, elute by salt or pH change...). Don't rule it out completely.
If you already have aggregates, adding NaCl might help prevent them...., or not. It depends on the amount (you can have "salting in" or "salting out" processes). In any case, if already formed, it might not help in dissolving them. And SEC is sensitive to highly hydrophobic species or conditions, where the protein can interact with the matrix.
With the information you give, as suggested by Tres, I would give IEX a second thought (after exploring what the pI of the protein is to choose the right matrix and buffer), depending on the composition of the elution fraction from your first purification step (pH and ionic strength).
Hi Sravya
Size exclusion is not generally a method of choice early in a purification method. To be preparative, you need large columns and resolution is easily compromised. Gel filtration as a means to desalt or change buffer on the other hand is extremely useful throughout purification.
A word of general caution. New column media can produce very low yields during the first few runs. There is a certain amount of non-specific binding. Your mileage may very.
As you are aware, the elution is related to shape, which if globular, is related to size. Aggregation is not uncommon and in some cases can be modulated with buffer conditions. I assume you are quantifying your protein with an accurate extinction coefficient and are calculating yields correctly. Are you monitoring fractions by hand or using a flow through monitor? Does the UV system measure at the wavelength your protein absorbs? I have seen many different kinds of mistakes made here. Just asking...
If there is aggregation, there is the possibility that the protein is stuck on the column. Do you see reduced flow properties or higher back pressure?
As you have a his-tag, you should have 80-90+% pure protein - if you follow the tag and column instructions. How do you assess purity?
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