How to prevent proteolytic degradation

[Choe Woo-seok]

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

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 Woo-seok Choe
 Dept. of Chemical Engineering
 University of Cambridge
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