Expression problems

WICKHAM,JOHN jwickham at aardvark.ucs.uoknor.edu
Sun Oct 16 00:11:00 EST 1994


In article <146087426wnr at genesys.demon.co.uk>, Duncan at genesys.demon.co.uk writes...
>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. 

IMHO, cells would express proteins best while they remain in exponential
growth. My protocol involves inducing expression with IPTG at an optical
density (595nm) of 0.6, then harvesting after 2 hours. The idea is to insure
cells are harvested prior to reaching stationary phase. Don't forget that
individual strains and media will vary in their growth curve (I use TG1 cells
and LB). You might want to do a growth curve if you plan on making more use of
your expression system, although OD595 at 0.6 is a good estimate of exponential
growth.

Hope this helps.
J Quyen Wickham
Univ. of Oklahoma Health Sciences Center> 
>Any and evry thought welcome.
> 
>Duncan      
> 
>-----------------------------------------------------------------------------
>Duncan Clark                        | Internet:    duncan at genesys.demon.co.uk
>G4ELJ                               | Compuserve:  100015.1406 at compuserve.com
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