Hi guys, I would like to know if there is any recommended websites that I can rely on for isoelectric point estimation? I used the one by expasy and it seems that the value deviates by at least 0.5 from the database where I retrieved the sequence. As such, I am not sure which is the one should I be following.
Also, is there any general rule on the pH to be used during protein purification? I learnt that it is usually one pH unit from its PI but does it matter if it is above or below the PI? Though I know that that will affect the overall charge of the protein, I am not sure if the pH I use will affect purification via the Ni-His approach.Thanks.
Hi Adam,
Yes, you can do isoelectric focussing using ampholines (gel) of different pH ranges in order to get the correct value of the isoelectric point (pI). If you want to use it for purification purpose using ion exchange chromatography, you can nuse the approximate value as well. Remember if pH of the buffer is lesser than pI, protein will acquire positive charge due to protonation of the ionizable groups (carboxyl - alpha, beta and gamma or amino - alpha, epsilon, imidazole and guanidino). On the other hand if pH of the buffer is higher than pI , protein will bear negative charge due to deprotonation of these groups. In order to get complete protonation/ deprotonation of these ionizable groups, pH should be 2 units lower or higher than pI of the protein. Therefore, you can generate either net positive charge or net negative charge on the protein and select weak cation exchanger (CM-Sephadex) if protein is positively charged or weak anion exchanger (DEAE-Sephadex) if protein is negatively charged for efficient binding of your protein to the ion exchanger. For the elution of your protein from the column, you can use either pH gradient or linear ionic strength gradient.
Let us hope that you may get your protein in active form and in sufficient amount through affinity chromatography.
Dear Adam,
It is true that you should keep you protein in pH higher or lower than your protein's pI. Proteins in pH= pI tend to precipitate due to the absence of net charge on the protein surface.
Expasy pI tool is quite reliable. Protein pI calculation is not so trivial, especially based only on sequence. If you need it only for setting up purification conditions pI calculated this way will work.
If you are planning to purify you protein on Ni-NTA you will have to keep pH above 7. The best will be 7.6-8.0. Low pH is causing histidine to protonate and to loose affinity towards resin. pH will also affect other protein purification steps like ion exchange.
Dear Adam,
The pI should be calculated from the residues exposed to the solvent, so only surface residues and those in accessible solvent channels should contribute to this calculation. However, this can only be achieved by knowing the 3D structure of a protein and for most proteins there are no structural data. As a result the pI can only be calculated/estimated from the protein sequence, which is fairly accurate nonetheless. Protparam from expasy will generally do a very good job and the pI it gives you can be relied upon for deciding what pH would suit your protein best.
Choose a pH value that is at least 1 point away from your protein's pI, though 1.5 to 2 points is preferable to make sure your protein is properly charged. The choice of going up or down on the pH scale depends on what you want to do with your protein. As Dawid says, if you're going to use Ni-NTA purification and your protein has a pI = 6, then go for pH = 8, it would make no sense to go for pH = 7 or, even worse, pH = 4 to 5. When you don't have to choose a pH based on a technique that requires a certain pH range, then choose to go either up or down the pH scale towards the region closest to pH = 7 to 8 rather than towards the extreme ends of the pH scale (e.g. with pI = 8 go towards pH 6 to 7 rather than pH 9 to 10).
I hope this helped.
Dear Dawid and Antonio, thank you all for the replies. They have been really helpful. The protein that I am purifying will be used to test its activity so perhaps changing the pH by too much may result in the loss of its activity. Currently, the problem that is most pressing is the low yield upon purification, which I suspect is due to the previously used pH being too closed to the protein PI. Hence, I will work on the pH for now. Thank you once again.
Is that a glycoprotein? The PI calculated from the amino acid sequence can be far away from the actual PI, if you have charged sugars (i.e. sialic acids).
You want to operate away from the PI to avoid aggregation. When the protein molecules become neutralized, they will lose one of the forces that repel them from one another, and may aggregate on you more easily.
Dear John, it is not a glycoprotein but it can be found in both the membrane and cytosol. Thanks.
If you have a low yield from protein purification you might not only want to try varying the pH, but also varying the buffer(s) that is/are used. I have previously worked with a protein complex that was only stable in a few of the commonly used buffers. Good luck!
Hi Josy, I am currently using PBS. Could you elaborate on what sort of buffer to use? Do you mean there are protein specific ones? Thanks.
I just meant that there are a lot of different buffers: TRIS, KPi, and MES, HEPES, MOPS (the last 3 are part of the Good's buffers designed for biochemical research) and that sometimes proteins are more stable if you try another buffer. It is difficult to predict which buffers are good for your protein, but you might want to try a couple of the once I mentioned to see if they give you better results.
You could determine the experimental pI in a Bio Rad mini protean for example. There is a precast gel which you can use for this kind of determinations.
I think this is the best manner to choose the appropriate way for purification your protein by ion interchange chromatography.
The technique to know the pI is isoelectric focusing. Either you buy the right precast gels to do the electrofocusing (similar to electrophoresis) or you may find a company that does it for you. If you do it yourself either you know approx the pI or you will have to use different gels with different pH ranges and going from a gel with a wide range to another with a narrow range to be precise in pI determination.
Knowing the pI is very important if you plan to use ionic exchange chromatography for purifying your protein. Depending on the solid phase (cationic or anionic) you may play around using a pH over the pI to have yuour protein charged negatively or under the pI to have your protein charged positively. If the solid phase is + and your protein is + will not be retained; if you change the pH and your protein is - it will be retained in the chromatography column.
Hi Sebastian and Rafael, isoelectric focusing is indeed one possibility. I am doing affinity chromatography via the Ni-His approach for now. Will be attempting these techniques that you guys mentioned if the optimisation of pH or buffer conditions still do not provide any improvement to the yield of my target protein. Nonetheless, thank you for the discussion.
Hi Adam,
Yes, you can do isoelectric focussing using ampholines (gel) of different pH ranges in order to get the correct value of the isoelectric point (pI). If you want to use it for purification purpose using ion exchange chromatography, you can nuse the approximate value as well. Remember if pH of the buffer is lesser than pI, protein will acquire positive charge due to protonation of the ionizable groups (carboxyl - alpha, beta and gamma or amino - alpha, epsilon, imidazole and guanidino). On the other hand if pH of the buffer is higher than pI , protein will bear negative charge due to deprotonation of these groups. In order to get complete protonation/ deprotonation of these ionizable groups, pH should be 2 units lower or higher than pI of the protein. Therefore, you can generate either net positive charge or net negative charge on the protein and select weak cation exchanger (CM-Sephadex) if protein is positively charged or weak anion exchanger (DEAE-Sephadex) if protein is negatively charged for efficient binding of your protein to the ion exchanger. For the elution of your protein from the column, you can use either pH gradient or linear ionic strength gradient.
Let us hope that you may get your protein in active form and in sufficient amount through affinity chromatography.
Hi Saad, thank you for the in depth elaborations. I am currently applying a pH that is about 1-1.5 units higher than the PI of my target protein. Thus far, the yield is pretty good and I am hoping it does not precipitate upon dialysis as I remove the imidazole.
You can use ExPasy online tool to calculate protein isoelectric point. Amiono acid sequnce of protein , you can find from Uniport. To purify protein by HIS/GST tag , its very important to adjust pH according to PI.
link:
http://web.expasy.org/compute_pi/
http://www.uniprot.org/
http://web.expasy.org/compute_pi/
http://www.uniprot.org/
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