insoluble protein in sf9(but soluble in mammalian cells)

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grace
grace's picture
insoluble protein in sf9(but soluble in mammalian cells)

Hi,
Recently, i've been trying to purify my protein of interest. The protein size is approximately 160KDa and i can purify this in mammalian cells by using Flag tag system. Now, I wanted to get enough proteins.. so I used bac to bac system. However, I can see my protein of interest only in insoluble fraction.. I've tried many detergents such as 1% Triton X-100, 1% OG and 1% cholic acid but still I cannot solublize this protein.
The thing I cannot understand is that I can solublize this protein in mammalian cells overexpressing this but i couldn't get this in sf9 cells.
This protein is an enzyme so I'm afraid to use Gn-HCl.. it'll be okay to purify an enzyme?
Is there anybody knows about this? and any solution?
If anyone gives solutions and answers about this, it'd be great help to me.
Thanks.

Sami Tuomivaara
Sami Tuomivaara's picture
grace,

grace,

First thing that comes to my mind is that the post-translational modifications on your protein (especially
complex glycosylations) are a bit differend depending on your expression system, here mammalian versus insect cells. Your protein might be very finicky about its glycosylations, and it is soluble only with correct glycosylation pattern. Invitrogen has modified sf9 cell line (called "Mimic") that do more mammal-like glycosylation.

Cheers,

Add colour 2 ur life
Add colour 2 ur life's picture
Hi,

Hi,
Here are  the some possibilities:
1.  Lysis was not complete.
2. Protein might get aggregated
3. Glycosylation pattern

So, if you want to do unfolding using GuCl for your enzyme (refolding as well).I dont think it will be a problem.

All the best

grace
grace's picture
Thanks for the replys:)

Thanks for the replys:)
I checked lysis conditions and it seemed to be okay because it worked well in control cells which do not overexpress my protein of interest.
Now, i also think the reason came from glycosylation pattern.
After testing several things, i'd like to test mimic sf9 cells as Suola mentioned and use Gn-HCl..
I'd really appreciate all the kind answers.
Thanks

kji
kji's picture
@ Grace,

@ Grace,

I am having the same issue of my 130KDa protein becoming insoluble in SF9s. But, it is soluble in mammalian system. So, I wanted to check if you had any luck with the Sf9 "mimic" or the GdnHCl?