Protein Denaturation

M. Singleton m.r.singleton at exeter.ac.uk
Wed Sep 18 10:30:43 EST 1996


Help !!
I'm trying to over-express a hydrolase-type enzyme from an extreme 
thermophile source in E. Coli. The recombinant enzyme seems to be 
expressed well, with high activity in the E. coli cell extracts. The 
problem is, that the activity "goes off" very rapidly (24 hours) even 
at 4°C in fairly highly purified samples. SDS-PAGE does not show any 
degradation of the protein, so proteolysis does not seem to be a 
problem. I've tried additives such as glycerol, DTT, EDTA, high salt 
e.t.c., but to no avail. The enzyme does seem to be very stable in 
crude extracts from the native organism however. Any ideas as to what 
is going on would be welcome.



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