Recently I've been purifying a 85kD protein from Rosetta. The yield is pretty good and the 260/280 is around 0.7-0.8. However, when I run this sample on FPLC superose 6, it showed very huge aggregation peak which is not consistent with a similar protein purified from insect cells(usually monomer). I used the same purification procedures. Does anyone have similar experiences? I used pET15b vector and used 1mM IPTG to induce at 17C overnight. Should I try to decrease IPTG concentration and decrease induction time? I've attached the SDS-PAGE and FPLC profile with this.
Hello Lan,
sounds like E.coli is not the proper host to express your protein. You may try E.coli with added shaperones or stick to eukaryotic expression systems. Alternative explanation, is that the purification procedures are not entirely identical, for example you may have heated E.coli lysate while sonicating
Is your protein rich of cysteines? or it is reported to be subjected to specific post-transaltional modification during biogenensis the e.coli is not able to perform?
Are you expressing the same sequence (including tag) reported for eucariotick expression?
Aggregation at high MW, as suggested by Andrey seems to be due to not correct protein folding.
1mM IPTG overnight is quite a lot. I would reduce this to 0.2 for O/N expression and reduce temp to 13C.
Have you ran an expression profile sampling the culture at different time points to see if the protein is being incorrectly folded from the start or if you are just not giving the cells enough time to fold properly?
Hi everyone,
Thanks for your suggestions. I've tried to rerun the sample from the soluble aggregate fraction, but unexpectedly it's quite stable and monodispersed from SEC-MALS although the peak is still very wide(4mL). I'll try to decrease IPTG amount and see how it goes. Unfortunately it doesn't seem to have any monomer and we don't have a way to test its activity in vitro.
Hi,
I would expect the host cell protein to be very different, Rosetta vs insect cells (e.coli vs euk.
I believe that you need to look into the purification of your target protein and change
to be effective to remove the host cell proteins from Rosetta. How do you know that it is aggregates that you see in SEC, could it just be host cell proteins that you do not get rid of with your method "optimized" for purification of your target protein from insect cells?
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