Problem with Induction (inclusion bodies?) - (Dec/23/2009 )
I'm trying to induce a fusion protein from Rosetta2 cells: ~12kDa protein attached to ~27 kDa YFP. I verified that my gene is in the correct orientation within my plasmid. Problem is that when I induce with IPTG, which I tried at 1 and .1 mM @ 37C, I get cessation of bacterial growth (which tells me that the load is being put on creating my protein) but I don't see my protein on a SDS-PAGE gel (coomasie blue stained). My protein of interest is associated with the membrane, so I tried inducing at OD600's of .6 and 1.3. I tried lysing my cells with up to 5% SDS hoping this would unfold the inclusion bodies but to no avail. I'm currently attempting to lyse with B-PER w/ lysosome to help purify what I think to be an inclusion body and will run a SDS-PAGE gel next monday. If this doesn't show any results, does anyone have any suggestions on how I should carry out my induction?
Grow the cells to OD 0.5 or so and then induce, wait 1-2 hours and then harvest the cells. You could also try growing at 30C or lower rather than at 37.
phage434 on Dec 24 2009, 08:32 AM said:
yeah, u might want to test it at 18C...also, maybe u should also check the uninduced culture....sometimes foreign protein expression may occur w/o induction.........
trying the uninduced culture is a good idea because you will get basal expression anyway ...and this "low" level expression will be better for soluble protein.
But it could be due to the protein itself ...some proteins tend to form aggregates. I once stumbled over a paper in whom the used 5% of ethanol in their cultures to promote expression of chaperones and thereby raised the amount of soluble protein. If you want to i can try to dig it out and send it to you!
Regards,
p
pDNA on Dec 25 2009, 03:47 PM said:
But it could be due to the protein itself ...some proteins tend to form aggregates. I once stumbled over a paper in whom the used 5% of ethanol in their cultures to promote expression of chaperones and thereby raised the amount of soluble protein. If you want to i can try to dig it out and send it to you!
Regards,
p
Hey P, that seems interesting. If possible, can you send that paper to me?????