Expression problems

Duncan Clark Duncan at genesys.demon.co.uk
Tue Oct 11 11:23:32 EST 1994


Hi Folks,

I have an interesting problem upon which any advice would be appreciated.

I have a set of 'identical' genes from different strains within a 
genus that are expressed in a pUC18 derivative. They derivative is a 
tac promoter instead of lac plus laciq in basically pUC18. Expression 
is quite reasonable (a few %) but IPTG has to be present continually and
I only get decent expression upon scale up when one uses innoculums of 
0.005% ie 1ml into 20 litres ie a certain number of doublings is required
in order to get a build up of product. The codon usage is definitely v.poor
and the gene product doesn't appear toxic ie growth with and without 
induction is similar. Different E.coli strains didn't make any difference to
expression. 

So I inactivated laciq and looked at expression in various lac+ and 
lac- E.coli's 'cos the xpression should now be permanently switched on.
This is the case and LE392 (lac-) looked very good for all clones 
on 3ml O/N cultures. However upon scaling up I'm back to poor expression
again. I've dropped the temperature down to 30C to slow the growth up a 
little and this helps. So my thoughts are that the only way to get high 
expression is to have a minimum no. of doublings before stationary phase is
reached, if that makes sense. Can anyone explain this? Is it due to codon
usage problems. Protease-, lac- E.coli doesn't help so I don't think 
proteolysis is the problem. One odd thing I do see is that I get 
better expression in any lac+ E.coli without IPTG than with. It is 
still worse than a lac- E.coli but odd all the same. 

Any and evry thought welcome.

Duncan      

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Duncan Clark                        | Internet:    duncan at genesys.demon.co.uk
G4ELJ                               | Compuserve:  100015.1406 at compuserve.com
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