How to prevent proteolytic degradation

Choe Woo-seok wsc22 at
Mon Nov 6 12:43:55 EST 2000

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
Many thanks.

 Woo-seok Choe
 Dept. of Chemical Engineering
 University of Cambridge
 Pembroke Street
 Cambridge CB2 3RA, UK
 Phone : +44 01223 740 979 (Home)
              +44 01223 334 786 (Office)
 Fax     : +44 01223 334 796
 E-mail : wsc22 at

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