You would have to explain what "submerged culture" means.

Secreted proteins are going to be in the liquid that the cells are growing in, so you need to remove the liquid from the cells without disturbing (lysing or stressing) them. You can do this by centrifugation to pellet the cells and remove the supernatant containing the secreted proteins or you set up a continuous flow system where growth medium is pumped into the chamber that the cells are growing in and allowed to flow out to a collection container. As the medium flows out, it carries the secreted protein. it is essentially a continuous culture system.

The flow through medium can then be concentrated using ultrafiltration or crossflow filtration and the proteins analysed. As long as you don't make the protein too concentrated, they should stay in solution and in their native form. However, there will be proteases present as well, so maybe some inhibitors are an appropriate addition.

Hi Matthew,

thanks for your suggestions.

For submerged culture, I mean fungal liquid culture. I'm gonna use centrifugation for mycelium separation from the broth; my bigger concern is about precipitation technique for sample concentration and cleaning up (I cannot use dialysis).

Can salting out (precipation in ammonium solfate) denature proteins in my samples?

The buffer I use for protein resuspension should include protease inhibitors or not?

In case it includes them, can they affect the "activity" of my enzymes?

Thanks in advance!

LB

Hi Linda,

the salting out should work. Be sure that the protein concentration isn't to high. I think you should use protease inhibitors. Will they affect your enzyme? Well, it depends on what kind of enzyme you are working with. In general they are quite selective and should not affect other proteins.

Be also sure that the temperature that you are culturing your microbes won't couse the denaturation of your enzyme.

Inhibitors are normally broad in their range of targets, so avoid the cocktail tablets and add specific inhibitors for specific classes or types of proteases.

I haven't tried salting out for years as ultrafiltration works really well (and the majority of my work doesn't require activity). Why can't you use dialysis? You would need to to remove the salt after salting out.

Hi guys,

thanks again for your replies.

Matthew, please, can you suggest me a device for ultrafiltration? After salting out, I'm gonna use Microcon, in case I need to remove salts.

According to your experience, does ultrafiltration preserve enzyme activities?