problem in protein expression

David-Paul Minde via methods%40net.bio.net (by davidminde from gmail.com)
Fri Jan 8 15:48:23 EST 2010


if NO expression, it may mean that your tag is useless for your target protein.
Some of the most useful(i.e. expression-enhancing) tags are SUMO,
NusA, MBP and GST.
Also tag suitability depends on temperature range which has been
recently demonstrated in a proteomic expression screen (PMID: 18226205
[PubMed] ).
I do the initial screening only with autoinduction @"tag-reasonable
temperature". Much simpler : 1 time series per construct.
Once you have good (=solubly expressing) constructs you can still go
back to (in some cases faster or more economic) IPTG-based expression.
cheers,
David


2010/1/8 Kyle Legate <legatekBLAH from hotmail.com>:
> ms wrote:
>>
>> hi all,
>> i am doing standardisation for protein expression and for this i am
>> taking 50 ml culture for induced and 50 ml for uninduced and earlier i
>> used .5mM IPTG conc. but i didnot see any expression. this i did twice
>> and also after IPTG induction i am keeping it at 18 degree for 12
>> hours and also the lysis buffer i am adding is 50mM Tris and 1mM EDTA
>> and i am not adding nacl but in wash buffer i am adding 150mM
>> NACLshould i add this in my lysis buffer and this time i am using 1mM
>> IPTG concentration and i am growing my culture at both 37 degrees and
>> 18 degrees when the OD of my secondary reaches 0.4-0.5, earlier i was
>> inoculating secondary at the OD of 0.3.but this time i have changed
>> this also. actually the earlier condition i was using for the full
>> length but this is the same protein but with 18 residues deleted at
>> the c terminal so i thought i have to change the conditions also
>> because earlier conditions which i used didnot work .so please suggest
>> me so that i woulb be able to get the expression and i can start my
>> purification.
>> thanks
>> ms
>
> You're doing too much work for a simple expression screen. This is what I
> would do to establish some key conditions, specifically an IPTG
> concentration course and an induction time course (doing it at different
> temperatures as well will set 3 parameters):
>
> Grow a seed culture to log phase but do not let it saturate. Set up a bunch
> of tubes with 0.8 ml of medium and seed each with 0.2 ml of your seed
> culture. Monitor one of the cultures for OD and induce when it's firmly in
> log phase, around 0.6-0.8. Induce with a range of IPTG concentrations (0.5
> mM should be the highest concentration, I have had best results with much
> less), and incubate for various time points, say 1 hour, 2, 4, 6, 10,
> overnight. At the conclusion of each time point, spin down the whole culture
> in an eppi and lyse the cells in SDS load buffer. Run the whole sample on a
> gel. Take the best condition and scale it up.
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David Minde MSc (TUM)

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