GST fusion protein purification - (Aug/31/2012 )

Hi all,
I am purifying GST fusion protein but i didn't get fusion protein. After IPTG induction my protein expressed well. i got good band with target size..but after purify fusion protein, i got vector size only in elution..There was no fusion protein band. I used Glutathione HiCap Matrix Kit system..i dont know where my fusion protein went..pls give me some suggestion...


I request you to please help me..

Looking for your kind reply..

Thanks in Advance...

With Kind Regards
VINOD

My overall conclusion to what you are describing here is that your protein is not soluble.

There are several possibilities:
1) what you see as good size protein after induction is in fact another protein that also is overexpressed when cells are induced e.g. a chaperone that is induced because your protein aggregates and the response of the cell is to produce chaperones not to have protein aggregates. In order to check for this I usually enrich my protein lysate in my protein by incubating 2 mL of cell lysate (after cell disruption and centrifugation) with 20-50 uL of glutathione beads; followed by 2-5 washes at lowest centrifugation speed. Like this you will not have to do a full purification before you know that you have only free GST. But be cautious: there are chaperones that bind to glutathione beads without having a GST -tag i.e. unspecific binding. But anyhow you would have the same chaperones present after the affinity purification step as well.
2) you do have your protein but it is found in inclusion bodies that are not in the cell lysate that you use for purification. This is why I always keep for an SDS-PAGE gel a sample of the pellet, clarified cell lysate and also this enriched fraction I mentioned above. Like this you know where you have your protein, if you have it at all. To be sure, I also do a WB against the GST tag (or the protein I am trying to express if I have a good antibody against it). Like this I would not confuse my protein with another protein that coincidentally has the same size.
3) your protein precipitates during the purification. This is why I do all my purifications in the cold room. Moreover, there is an experiment, called thermofluor through which you can screen several buffers in which your protein has increased stability. If you have such a buffer, your protein would not precipitate during purification. However, if you don't have this experiment established in your lab, it is a bit difficult to do. You basically need SYPRO ORANGE and a RT-PCR cycler for it, besides the commercial buffer screen (which is not at all expensive and you can use it for several years)

I cannot give you an advice about what to do to get your protein until you do some of the things above for us to see where exactly your protein disappears.

Andreea

Thank you for your reply...