protein expression - How to express protein of a high molecul (Aug/07/2004 )

Dear all,

I will hope to express virus protein with a high Molecular weight about 186kD. Somebody once tried to express via E.Coli system, but failed to do so. We hope to express this protein and make it crystallization. Would you be so kindly to give me some advice about this experiment, such as which expression system shall be used, and someone said the high MW protein is more easily to aggregate, thus how to avoid this disadvantagement.

Thanks in advance!

Seagull518

What sort of virus protein? Does it have any glycosylation sites? Disulfide bonds? Is it internal in the virus and therefore devoid of the above two?

Thanks for your reply.
It is a RDRP in one plant virus. Do you think glycosylation or disulfide bond will affect the protrein's expression.
We have tried the cell free system for translation in vitro, and the result revealed it worked well.
Do you think some protein can not be expressed in E.Coli at all.
Thanks!

One of the problems with expressing genes of higher-level organisms in E. coli is post translational modification. This means that you might get less expression in E. coli, but you also might get better expression because the E. coli system is "simpler" than most others. I would recommend cloning the gene in a common expression vector, like for example pET which utilises the very strong (and almost completely silent in the absence of IPTG) T7 promoter, in BL21 cells and see what happens.

Good luck

Thanks for your reply. I will try the protocol you said.