Dear Ravish

there are many reason because a recombinant enzyme can be not active and to hypotize it is important to know the specific sequence properties of the protein.

for example:

- e.coli is not able to introduce post translational modification as glycosilation and may fail to correctly form S-S binds which are essential for stability and function l of some Protein.

- moreover if your protein have to contain a cofactor as for example a metal la it possible that is not present after protein purification and you have to add it to the protein to reactivete it

- on the contrary is it also possible that during purification step (eh during the imac) some metal from the coloum bind thr protein aspecifically and may inhibit its function.

So to undertand where is the problem you need to anase well giù protein sequence or literature and perform on your purified sample some b

biochemical quality controll as for example:

- size esclusion chromatography to check aggregation state

- sds reducing and not reducing if it have to contain S-S bonds;

- dsf (thermofluor) or circular dichroism of your protein with and with-out cofactor ( eg metals ) or edta for example and at the end undertand if you need to add some step on sample preparation or go back to protein expression by changing condiction or host.

about stability and purification in cold room. the most proteins (an reception are the proteases) can be purified at rt adding proteases inhibitors and stored from short time at 4C. Long terms storage at -20 or -80 can require addiction of glycerol to prevent protein unfolding and aggregatiin bur it depends protein to protein.

cell lisys if performed with mechanical appriach as sonication can be a crictical step where you can induce protein unfolding of you use a to strong cicle and temperature of you sample increase to much during the cicle.

good luck

Manuele

Hello Ravish, as Manuele said, but also check the sequence of the plasmid in case an unwanted mutation occurred that is inhibiting protein function. Good luck,

Hi there,

Have you got a positive control for your glucosidase assay just to check the assay is OK? Is this assay specific/suitable for your glycosidase?

If it is a matter of protein unstability during purification, you should be able to detect at first the increased glucosidase activity in the whole cell lysate in which the protein has been expressed (compared to the one detected in a cell lysate with no expression).

An other more problematic issue might be that your protein is not active from the start (due to misfolding and/or the lack of something activating the enzyme)...

Hi Ravish,

There are many factors that affect protein activity, but the key question is whether you really know your target protein? I think the temperature you mentioned when purifying has an effect on protein activity, but it is not the most important.

1. Please carefully check whether the cloning is correct. Have you introduced improper mutations? Does the codon need to be optimized?

2. Is the expression system used appropriate? Do you need to use chaperones or add metal ions that help your protein of interest remain active?

Good luck!