Baculovirus Expert Needed (Occlusion bodies) - Can proteins become stuck in occlusion bodies in baculovirus? (Jan/12/2011 )

Hi,

I'm trying to express a series of rather large misbehaving proteins in Baculovirus. The proteins are His-tagged and would like to purify them with affinity purification. The purification step has not worked a couple of times, but we do see what we think is the protein when we run a total cell lysate. I was wondering if the protein can get stuck in insoluble occlusion bodies like in E.Coli? If they can is there any way to get the protein out of the insoluble portion without strong denaturants followed by refolding?

Thanks.

would enjoy any comments or suggestions even if they do not directly answer the questions posed.

CW

what cell line do you use? Sf9? ...the protein for sure get stuck in the golgi for example ...then you would see it in the whole cell lysate but not in the culture supernatant.

Regards,
p

pDNA on Wed Jan 12 19:50:56 2011 said:

Thanks,

Yes using SF9. Is it common for recombinant proteins to get stuck in the golgi? is there anyway to get them out without heavy denaturaunt?

Hola, to me, the character of soluble or insoluble is due to the properties of the molecule.Knowing the sequence, there are tools in internet which predicts the % of probabilities of solubility and other which helps to calculate the isoelectric point.So, if the solubility prediction is medium-low, knowing the isoelectric point, I´ll try modify pH, composition of buffer , reducer and light detergent under Ni-agarose compatibilities in order to have low levels of soluble protein, after that, there are intermediate concentration of urea wich helps to solubilize without total disfolding, but each protein is a world and all of this couldn´t run. Buena suerte!

i do not really get your problem? ...are you trying to secrete your protein in the supernatant? ...you are just finding it in the whole cell lysate and not in your supernatant? ...therefore you conclude that is trapped in inclusion bodies? ...right?

Regards,
p

pDNA on Thu Jan 13 07:37:42 2011 said:

Yes trying secrete my protein in the supernatant. Ive used this expression system 3 times before resulting in enough protein for crystallization. My question was as basic as can proteins get caught up in inclusion bodies in this system like in Ecoli? I'm not interested in obtaining structures from proteins that have been removed from inclusion bodies with strong denaturants and than refolded because of increasing evidence that such structures are often not the native structure found in the cell due to the abscence of co-translational foldIng.

Thanks.
CW

protolder on Thu Jan 13 07:02:09 2011 said: