If E.coli does not like AK (i.e. it is slightly toxic to
the cells or perhaps it doesn't like using up so much
energy to express it), then some mutation may be selected
for that prevents high expression. I would be tempted to
try starting from the very beginning with fresh plasmid
containing your gene of interest. Ideally, this plasmid
will not have "lived" inside an E.coli cell too long and so
won't have been altered itself. Often, in expression
systems, it is recommended to re-transform the E.coli that
you will be using for your expression every time you get
ready to grow up a batch. All the best.
John Marcus
> 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.
>>
_________________________________________________________
John Marcus Marcus at tpp.uq.edu.au (Dr J.Marcus)
Cooperative Research Centre for Tropical Plant Pathology
5th Level John Hines Building
University of Queensland
St. Lucia, QLD 4072
AUSTRALIA
Fax: 61-7-3365-4771
Phone: 61-7-3365-4764
