Dear All,
My protein is getting expressed in undouble form. I want to purify it in native form so I have not attached any tag. Can any one has experience and guide on how to purify protein from pellet having no tag?
You can purify proteins natively even when they are tagged - in fact, this is the method that allows for the highest yield and purity of native proteins. His-Tags are frequently used for this, as well as GST tags, which act both as an affinity and solubility-promoting tag.
If you really, really need to purify your protein from the pellet because you do not wish to include any tags, you will have to unfold and re-fold your protein. For unfolding, you can use a high molar concentration of Urea-containing buffer and solubilise the protein that way. Dialysis back into a native, Urea-free buffer is then needed to refold the protein. But this has many drawbacks - your protein may not re-fold correctly, many chromatographic techniques are either affected or unusable in denaturing conditions, and the purification protocol becomes more complex.
My advice is: If you want native protein, try to determine a soluble construct by determining if the C- and N- termini of your protein are disordered and promote precipitation. You can do this either via putting the sequence into a disorder prediction software, or via limited proteolysis and mass spec. Then, try a variety of tags and cleavage sites to clone your insert into. Many plasmids nowadays even include cleavable tags that can be cut off using either 3C or TEV protease, leaving you with a tag-free protein after cleavage.
Hi Faiq,
Are you refering to methods to isolate your protein from the pellet? Have you performed SDS-PAGE or HPLC to separate the mix of proteins and western blot to identify it, in order to design an additional approach to purify it?
Refolding after solubilization may have resulted in a scrambled structure depending on your protein (see the anfinsen experiment). You may test the refolding kits to overcome the problem. Otherwise, considering the protein is in the cell lysis pellet (as inclusion body/membrane protein, etc.) and it is not precipitated by TCA or salting-out protocol, indeed you are going to have it denatured after solubilization by using urea/guanidine or detergent-based agents. If it is not a problem to go from intact protein rather than its native form, you can extract the protein from the gel by utilizing passive diffusion, electroelution, or by simply gel dissolving. In all cases, your gain will be low due to the loading limit of the page and further purification may be needed. Therefore after solubilization (by employing chaotropes) you may test to purify your intact protein by conducting one of the IEX/HIC/SEC techniques or using them as a tandem. If gel-based is your only choice, I would suggest adding chromotafocusing before SDS-PAGE, therefore using the 2D PAGE technique will give purer protein at inactive form.
I see two ways how you can approach your project: 1. You can in fact use an affinity tag for the purification, but remove it later on. Some expression vectors for the GST-tag even provide on their own protease cleavage sites between protein of interest and the GST-tag. You could use these vectors or copy that system to the affinity tag that you use.
This page explains the GST-tag example:
https://cube-biotech.com/gst-tag-how-a-protein-helps-to-purify-a-protein#removal 2. Use affinity chromatography (AC), but with a custom bead. AC uses the artificial added affinity of the tag towards whatever is on the bead. But you can also couple something to the bead that has a natural affinity to your protein of interest. Our favourite example here in my lab. To purify a neuro-receptor (ACHr) we coupled Neurotoxin-3 to agarose beads. The toxin has the natural affinity to the receptor and therefore we had an AC without any affinity tag. More on that here:
https://cube-biotech.com/customized-resins-maximum-specificity
I hope one of these options might help you.
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