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GST-fusion protein degrades in-vivo


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#1 Andrey

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Posted 18 May 2011 - 04:15 AM

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?

#2 ntgxw

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Posted 18 May 2011 - 03:07 PM

what is your bacterial strain?

#3 Andrey

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Posted 19 May 2011 - 02:14 AM

Normally I use BL21 Star (DE3), but I've also tried C43 and Lemo21 with the same result.

Edited by Andrey, 19 May 2011 - 02:15 AM.


#4 pDNA

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Posted 19 May 2011 - 11:57 AM

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!

Good luck!

Regards,
p




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