So I have an enzyme with pI 5.5. I'll try to produce it in Pichia in medium with pH 4.0 or 4.5 as it grows better. However, as I perform purification in basic pH (7-8), I want to change the pH of the solution. What is the best way?
As the protein has lowest solubility at pH = pI, I'd assume it's better to do it with diluted solution to avoid protein precipitation, but if I'll have e.g. 5 litres, that would be quite impractical. Also, is it better to do it quickly (e.g. add 1/10th volume of 1M Tris buffer) or slowly in dialysis (but can you imagine how long it had to be to accommodate 5l? ).
What are your recommendations?
If the protein is intracellular, you will spin down the cells from the growth medium and resuspend them in a cold buffer for extraction. This buffer will have the desired pH and composition for the purification.
If the protein is secreted into the growth medium, then after you spin down the cells you could add concentrated buffer such as Tris-HCl (pH 8) to raise the pH. This will also raise the ionic strength of the solution, which could be a problem only if your next step is to capture the protein with an ion-exchange resin.
Of course I mean extracellular expression, otherwise I would not bother.
So you're rather for the fast change.
Tomáš-
Fast is better than slow to avoid ppt at the pI point of your protein, which you unfortunately have to pass to get to pH 7-8. Don't know what your purification method is, so I can't say much more.
Also, you may want to do a small test run on conditioned medium (without your protein) to see if any of the proteins present in the medium precipitate during the pH increase. Otherwise you can go the old fashioned way and do an ammonium sulfate ppt curve to enrich for your protein, then bring it up in the purification buffer of choice.
Hi Tomáš, I agree with Becky in that it is much easier to precipitate your enzyme into a smaller volume, then you can bring up the pellet in physiological pH buffer, an/or do buffer exchange by dialysis.
You can use the low solubility of the enzyme at ~pH 5 to your advantage in precipitating it, either by ammonium sulfate, PEG, etc
If you don't want to go that route, then you could try using a very dilute buffer in your culture system at pH 4. Then you don't have to add a ton of buffer to your 5 L prep.
BTW, what is the buffer system you are using in your culture?
I was thinking about ammonium sulphate precipitation (although can you imagine the amount for again say 5L?), but my colleague was once told from our professor that there's no chance to preciipitate them because they are too diluted and they will not meet in the medium to settle down.
@Steingrimur: what do you mean with the dilute buffer at pH 4?
not dilution, but fermentation in 5l of media (well, now I did it actually in 10l)
Hi Tomáš, I was thinking that if you have a 5-10 mM pH 4 buffer in your culture, you can add a small amount of solid NaHCO3 to your prep to bring it up to pH 7.
However, you can still add solid bicarbonate to your current 5-10 L prep to get it up to pH 7.
But after that, you still have 5-10 L of media at pH 7. What do you plan to do with it.?
FYI Tomáš, I entered into a discussion on RG about how to reduce sample volumes before doing column chromatography.
https://www.researchgate.net/post/How_do_I_reduce_the_loading_time_of_protein_on_chromatography_columns
Granted, Hemanth has about 10-20x the volume you have, but there are ways to concentrate < 10 liter volumes on a non-industrial scale.
We have some system for concentrating large volumes, it takes 10 litres (plus 2 litres wash/buffer exchange) in something like two hours, so that's not such a big problem.
Great!, so fill up a truck with NaBicarb, dump it into your prep until the pH is 7 and concentrate.
(/sarc)
I think ammonium sulfate precipitation would be difficult with such a large volume, especially if the protein is dilute. Even if the protein precipitates, you would still have a huge volume to centrifuge, which is OK if you have a high-volume centrifuge. Concentration by ultrafiltration would take forever with the usual lab equipment, but could be done quickly with a tangential flow filtration unit. Capturing the protein from the bulk medium by adsorption onto a resin is another option.
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