GST-fusion protein degrades in-vivo - (May/18/2011 )
I'm trying to purify a membrane protein using the GST fusion tag. The plasmid is pGEx-4T-1. The expression is performed in autoinduction media at 18C. Howerever, I observe 2 bands on my Western blots corresponding to my protein+GST and GST alone. I.e. GST gets cleaved somehow in vivo. Does anybody have any clues how to avoid this?
what is your bacterial strain?
Normally I use BL21 Star (DE3), but I've also tried C43 and Lemo21 with the same result.
have observed this phenomenon several times ...GST can be a real pain!
some say it is due to unspecific cleavage of the thrombin cleavage site in E. coli ...you can try using different strains like e.g. JM108/109.
you can use 1% glucose in the medium to prevent leaky expression of the tac promoter, additionally just do a short induction (not more than 2-3h) and use a protease inhibitor after lysing your cells!