I am planning to purify resuscitation-promoting factors produced using an expression plasmid in an E. coli expression strain. The proteins inactivate shortly after purification.
I have explored techniques such as non-aqueous co-solvents and glycerol additives but was wondering if there is an optimal strategy to stabilise the protein for long term storage whilst retaining enzymatic activity.
You can always purify under denaturing conditions with urea or Guanidine HCl. The unfolded protein will be stable under these conditions for quite a long time. However, you must then find a way to refold the protein prior to using it, which is some cases is not difficult but in others it can be very difficult especially if your protein contains disulfide bonds. In addition, to purify under denaturing conditions you will need to use something like Ni- NTA, Talon or another affinity matrix that is functional in harsh conditions.
I think gycerol (10%) stabilizes protein for long time. my enzyme is as the same you. I can keep it active with glycerol for 2 mouth. it's so good to add your glycerol in elution buffer ( because protecting enzyme immediately from high concentration imidazole.
It is also important to remove imidazole immediately after purification by dialysis bag or centricon tube .
Regards
Hi Andrew!!
There are several strategies to store your protein in active form.
(1). After purification you can dialysed your protein in 50 mM phosphate,150 mM NaCl and 1mM betamerceptoethanol. Change the buffer 2-3 times while dialysis.
(2). You can use 50 mM phosphate,150 mM NaCl and 20% glycerol for dialysis. Make sure the pH of the buffer should not be near to your protein pI.
(3). After dialysis you can store your purified protein in 50% glycerol for long time. Before enzyme activity you again need to dialysed your protein (stored in 50% glycerol) in suitable buffer for your enzyme activity.
(4). You can optimize different buffer such as Tris, phosphate or PBS to get active protein. Change in buffer sometimes helps in above problem.
(5). you can also dialysed your protein in buffer containing 0.1% Triton-X 100 or Tween-20. Detergents may helps to maintain the protein in native form.
These mentioned condition you can try to get rid of your problem. hope it works.
Good Luck.
the one assumption that I forgot to mention is that you should store you protein as concentrated as possible, and if you cannot get it concentrated without precipitation, you can alwasys add a macromolecular crowding reagent- the simplest being BSA or a similar protein that will not interfere with your activity.
Besides the suitable buffer system and glycerol, you could try some additive such as sucrose, trehalose and betaine as stabilizer; L-arginine, glycine and Acetamide as aggregation inhibitor. High con. of glycerol may be necessary for long term storage, and lyophilization could be an alternative option.
This new review addresses aggregation and some other stability problems: http://www.ncbi.nlm.nih.gov/pubmed/24211444 (Lebendiker & Danieli)
A simple way is high salt precipitation. We have found that storing your protein as an ammonium-sulfate precipitated slurry is a simple way to stabilize some proteins. If you use this as a late purification step then you can keep your purified protein in this fashion with relatively good stability at 5 degrees C.
try avoiding gel filtration step till you actually are going to use the protein. in our experience gel filtration results in loss of co-factors which stabilize protein or activity. just do affinity [tagged protein] followed by rapid ion exchange. also set up a 96 well plate with different buffers and check protein for activity under different buffers.
you are sure to find the right buffer
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