purification of an unstable mutant

Zheng-Tao Shi ztshi at magnus.acs.ohio-state.edu
Mon Sep 27 23:03:18 EST 1993

I encounterd some difficulties in purifying a mutant enzyme. First of all, I 
lost a lot of the enzyme after breaking the cell even though I used PMSF. (The 
expression level of the mutant is comparable to that of WT and there is no
obvious loss for WT after cell lysis even without the addition of any protease
inhibitors). And this severe loss of the mutant seems to exit during the whole
purification procedure. Secondly, instead of being eluted within a narrow salt
gradient range from a P11 column (an anion exchange and phosphate affinity
column) like the WT, the mutant enzyme started coming off the column at very
low salt concentration (50 mM) and appeared in all of the fractions up to 700 
mM salt concentration; basically, I always got a very large and flat peak.
Finally, following the P11 column, a gel-filtration column did not give good
seperation either, which I suspect is due to the dirty viscous loading sample
resulted from the large amount of fractions of P11.  All these things added up 
and I barely got any pure enzyme at the end. Any suggestions on solving my
problem? Your help will be greatly appreciated.

Zheng-tao Shi
The Ohio State University
ztshi at magnus.acs.ohio-state.edu

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