How to prevent proteolytic degradation

ChenHA hzhen at freeuk.com
Thu Nov 9 19:13:57 EST 2000



Choe Woo-seok wrote:
> 
> 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?

Nothing approaching it.  Proteases working in 8M urea must be
unusual.  It should be noted that some membrane-associated proteases
are co-purified with inclusion body (I think OmpT is one), so use a
cell strain that is deficient in such proteases (you can use BL21 I
think, but can't remember if it produce such protease poorly or not at
all).  Add some EDTA as well if you are not using His-tag. 


> Many thanks.
> Woo-seok
> 
> --
> -------------------------------------------
>  Woo-seok Choe
>  Dept. of Chemical Engineering
>  University of Cambridge
>  Pembroke Street
>  Cambridge CB2 3RA, UK
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