How to prevent proteolytic degradation

Emir ekhatipo at NOSPAMmidway.uchicago.edu
Mon Nov 6 16:51:18 EST 2000


As far as I know, HMS174 expresses ompT and lon proteases which may degrade
the target protein during expression or further purification. Can you use
BL21, BLR, or B834 cells that are mutated to lack those proteases, or there
is a special reason you prefer HMS174?
Emir

"Choe Woo-seok" <wsc22 at cam.ac.uk> wrote in message
news:8u6qr1$cg4$1 at pegasus.csx.cam.ac.uk...
> Dear all,
> We have the inclusion body protein expressed from E. coli HMS174(DE3).
> When the IB protein is dissolved in 50 mM Tris, 50 mM DTT, 8M Urea, pH
8.0,
> the proteolytic
> degradation of target protein (Mw = 65 kd, with 6xHis tag) started.  We
> repeatedly saw four
> four major degradation products on the 12% gel, and the degradation rate
> looks quite fast.
>
> To prevent the degradation, we tried pH shift from 8 to 5, from 8 to 11.5,
> and also temperature
> shift from room temperature to 65'C for 1 hr and the addition of PMSF.
> Unfortunately, all efforts did not work at all.
> Is there anybody who has experienced the similar problem and sorted it out
> finally?
> Many thanks.
> Woo-seok
>
> --
> -------------------------------------------
>  Woo-seok Choe
>  Dept. of Chemical Engineering
>  University of Cambridge
>  Pembroke Street
>  Cambridge CB2 3RA, UK
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