Hello everyone,
I'm working in my doctoral thesis in the purification of a lipolytic enzyme from an halophile archea. Currently, I'm trying to purificate the recombinant enzyme using Haloferax volcanii as an expression system. The problem is that suddenly when filtering my crude extract through a 0.45 um nitrocellulose membrane I lose 90 % of the enzymatic activity, when that did not happen before and at most I lost 20 % of the activity.
Has the same thing happened to someone else? Or do you have any ideas that could help me? Thanks!
Hello, for more reliable answer you need to find out.
1. Is the enzymatic activity has some co factors which can be removed during fermentation.
2. Is the enzyme problem with size or structure of enzyme which can be changed during filtration.
3. Is the activity caused by living microorganisms in enzyme filtrate by removing them with filtration you are losing activity.
You can use other filtration technique like gel filtration, membrane dialyse, preparation HPLC etc.
Nitrocellulose binds proteins by hydrophobic interactions and is therefore unsuitable for your application. Use a non-protein binding membrane like polysulphone instead.
You may also use regenerated cellulose filters. Do not use PVDF or nitrocellulose material otherwise you probably will face the non-specific binding...
- If you use small syrgine filters, please use nylon or polysulphone or regenerated cellulose filter. Hydrophilic membrane made of regenerated cellulose exhibits low protein binding for sample preservation
- https://www.agilent.com/cs/library/applications/5991-1308EN.pdf
Thank you everyone for your answers!
Karen A. Darbinyan I forgot to mention that this happened with my lysate extract. Before the step of purification with an affinity column I filtered the extract through a nitrocellulose membrane and loss 90 % of the activity. But this never happen before, at most I lost 20 % of the activity. This was the first time that the enzymatic extract lost all the activity.
If you have more total protein in your sample you will procentually loose less. Does that explain your situation?
- Badges
- Science topic