Hints for protein export periplasmatic space during expression
hzhen at freeuk.com
Fri Sep 22 15:49:35 EST 2006
On 22 Sep 2006 12:05:31 -0700, "Breslauer" <breslauer1981 at gmail.com>
>Hints for protein export periplasmatic space during expression
>I would appreciate all ur posts here about hints u use or know how to
>determine such conditions in that the bacteria (I'm thinking about
>particulary 3 strains we have in our labs E.coli C600, E. coli
>DH5alpha, and BL21 with lambdaDE3) to produce soluble protein (I mean
>exported to the periplasm). We use different plasmids - all of them
>have specific signal sequence either specific for strain E.coli or for
>our protein (which comes from Salmonella Enteritidis strain). We search
>for optimal conditions for expression the protein and spedition to the
>periplasm, howether in all cases protein in 99% appear as inclusion
>bodies. Renaturation wa unsuccesful.
>So we heard about
>1 inducting the culture by addition IPTG very early (when OD600 = ~0,2)
>do u agree it is good idea?
>2. lowering the temperature after induction to 27C deg and let it grow
>in the presence of IPTG for 12 16h.
>But maybe there are more efficient tricks?
There is really no trick for proteins in the periplasmic space, it
would be easier for you to read some review articles on this.
Different proteins would behave differently, you may read some papers
where they make obscene amount of protein, but it may be useless for
your particular protein.
Howver, in general, if the main problem for you is the aggregation
into inclusion body, then any strategy that reduces the rate of
protein production may help. So lower level of IPTG (that may
depends on the cell strains and vector you used, but you can go as low
as 0.02 mM, experiment and see) . Lower temperature may also help,
some people express their protein at 16 deg C. Or perhaps choose a
vector that express at a lower level (i.e. weak promoter) rather than
one that does it at very high level.
Some people add additives such as glycine to their media, or do
osmotic shock by adding high salt and sorbitol concentration. You may
also coexpress other proteins, some people coexpress many different
proteins and you might consider getting plasmids from them. I'm
thinking of coexpressing some proteins as well for periplasmic
expression, but may not pursue it and will probably now express my
>I would appreciate all opinions
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