Why AK is not expressed consistantly?

Yan Lin iahd500 at ruby.iupui.edu
Wed Nov 12 23:01:05 EST 1997


Hi all,

I'm a graduate student doing biophysics with a stronger background in
physics than in bio-related areas.  Earlier this year we obtained an E.
coli strain (SMH 50) which overexpresses adenylate kinase (AK).  My very
first prep went very well - a yield of >70mg/L was obtained with
excellent enzyme activity.  But, ever since then I have not been able to
get consistant results in quantity and activity.  To make sure that the
bacteria from a "strong and healthy" origin are picked before large
scale amplification I've been measuring the ratio of Volume_Activity
(U/ml) and OD at 280nm for crude extract of each 5ml overnight
preculture.  This ratio varies in a wide range, eg. 5 ~ 100 U/OD.  What
troubles me is if a saved culture sample (plated on agar dish, or with
15% glycerol in freezer or liquid nitrogen) with high Activity/OD is
inoculated again the ratio may turn out to be much lower after overnight
shaking.  I'd like to know if this Activity/OD is the best measure of
the efficiency of protein expression in bacteria cells.  What are the
possible reasons for low and/or inconsistant expression?

Many thanks for your help.



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