Hello, I'm working with a glycoside hydrolase and I plan on freeze-drying my protein so that I can ship it some colleagues across the country. I have speed-vaced this protein suspended in 50 mM sodium maleate, pH 6.0 and when I re-suspended the protein in the same buffer it had severely reduced activity compared to a sample in solution that I didn't speedvac.
I've been looking into cryoprotectants to add to my solution before I speed vac my samples so that when we speed vac the samples and ship them out, my colleagues can re-suspend them and still have good activity. I've found that sucrose, trehalose, BSA, PEG and sorbitol are commonly used for this, however, I'm having difficulties finding specific concentrations or weights of these additives. Any info helps, thank you.
I think you mix to different methods here. Speed-vac is different from lyophilization (freeze-drying). Speed-vac is basically a centrifuge with mild vacuum and usually mild heating. It is used for drying down small volume, relatively heat-stable sample. Freeze-drying is a sublimation process used for larger heat sensitive samples. It uses high vacuum and low temperatures (−50 °C and −80 °C). You sample has to be frozen before placing it in the lyophilizator. Make sure you really use a freeze-dryer and not a speed-vac. Putting your protein into a speed-vac will cause degradation.
Thanks Cornelia,
I should state that I'm now working with a split version of a protein and I currently don't have access to a lypholizer, hence why I'm using a speed vac. I had speed vaced the full version of the protein and when I resuspened it in a 50 mM sodium maleate buffer I got around 80% of the original activity, so you can speed vac proteins and still get activity.
However, when I speed vac my split protiens and combine them I only get around 15% of the activity compared to the non-speed vaced form. I would like to boost this number closer to that 80% range.
So your split protein is more unstable then the full length version to the speed vac conditions and/or being highly concentrated. Instead of spending weeks finding optimal conditions (if there are some besides using a lyophilizer) why don't you send your protein on dry-ice? From what you said it is domestically. Considering the time and protein you might waste in optimizing, overnight shipping is the cheapest way.
I agree with Cornelia. If you use a speed-vac instead of a freeze-dryer, you will inevitably lose significant amounts of activity, as you have seen. I'm sure somebody at Calgary must have a lyophilizer you could try. However, if you really need to avoid freeze-drying, I would try a couple of different concentrations of cryoprotectant. The polymeric ones like PEG and dextran tend to work better at lower concentrations, but the low-MW ones like trehalose and sucrose are easier to remove. Use the lowest temperature you can get away with without increasing your drying time too much. We have used 1% - 2% dextran with some success as a stabilizer with our glycosyl hydrolases, but they have a specific dextran binding site, so I don't know if dextran would work as well for you.
I agree with Cornelia that it makes more sense to ship the frozen protein on dry ice than struggling to stabilize the protein during drying. If you take this approach, make sure you use enough dry ice to keep it all from evaporating during shipment.
If your material is to be shipped regularly you should look at freeze drying (your speed vac is unlikely to match the performance of a sophisticated freeze dyer) but it can take a lot of effort find the best conditions. You could start with 1-5% sucrose or 1-5% trehalose (final concentration), either of these work well for many proteins. Ideally you need a freeze dryer that allows control of shelf temperature, which generally is going to have to be -30 to -50 range. However, for a one-off shipment I agree with the others, dry ice is definitely the best solution.
Do you know if your protein is stable when it is simply frozen and thawed. It may be that the freezing is the problem not the drying. If it is the freezing send it express on wet ice in a thermos flask..
Good point from Peter - and exactly how something is frozen is important. A risk with slow freezing is that the solvent (i.e water usually) freezes from the outside of the tube. This can happen if you transfer a few mls of sample to a -70 for example. The buffer/salts become increasingly concentrated in the centre of the tube, which may denature your protein. A few microlitres of sample in the same freezer may give a different/better outcome. You can accelerate freezing if required by using dry ice/ethanol or dry ice/acetone (the solvents allow rapid heat transfer compared with dry ice alone) or even liquid nitrogen (but may sure your tubes can withstand this sort of treatment!).
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