Help needed for GST-fusion expression

ijiwaru at wheel.dcn.davis.ca.us ijiwaru at wheel.dcn.davis.ca.us
Wed Sep 25 14:50:30 EST 1996


In article <Pine.SUN.3.91.960925105529.10218A-100000 at newssun>, Jian Zhang
- Cell Bio <jzhang2 at newssun> wrote:

> Hi! everybody.  
> 
> I am trying to express a transcription factor as a GST fusion.  The 
> vector I used is pGEX-5-1.  First, I put the whole gene downstream of 
> GST.  I have no luck to see any expression of the fusion protein on 
> Coomassie stained gel.  I also did a western blot with GST antibody, 
> same result-- no protein.  Then I thought I should try different stains, 
> I tested common strains, such as DH5a, HB101, JM109, BL21,etc.  No luck 
> either.   
> 
> It seems the bacteria grow very slowly after I add IPTG.  So 
> my next guess is this fusion may be toxic to E.coli.   To this end, I 
> constructed another plasmid in which the C-terminal half (about 200 aa) 
> is attached to GST.  I cannot detect protein from this new fusion 
> either.  
> 
> All the constructs were confirmed by full-length sequencing.  I 
> cannot even see GST peptide from my fusion construts.  To confirm 
> that the GST and the vector sequence is still correct, I cut out my 
> insertion sequence and re-ligated, I can easily see GST expression.  
> 
> I hope somebody out there can come up ideas and help me out!
> 
> Many thanks in advance!
> 
> 
> Jian Zhang
> 
> My email:   jzhang2 at newssun.med.miami.edu
> 
>     

I can't remember off-hand what promoter drives the expression in pGEX-5-1,
but make sure you have it shut off as much as possible when you don't want
you're protein expressed.  With a Plac driven expression plasmid I have, I
have to use about 0.5% glucose to insure that I have minimal expression
even in the absence of IPTG.  There are two directions you can go in terms
of your expression conditions.  Check the amino acid sequence of your
protein and compare with an E. coli codon usage table.  If your protein is
full of "unfriendly" codons, try lowering the amount of IPTG and let your
expression go longer.  If you really think your protein is toxic, common
with many membrane proteins, grow your culture under repressed conditions
to mid to upper-mid log phase and then induce with high level IPTG for a
short period of time (2-4 hours).  If you're using a promoter under
control of lac operator, use a good lacIq strain.  From what many people
have said in this group, don't keep your expression cultures around for a
long time.  Once you start expressing, express a lot and then expect that
you will have to re-transform to get optimal expression later on.  I've
found with a couple of my constructs that I have about 3-4 weeks before I
have to re-transform.  Guess I'm just lucky.

Good luck,

Lyle Najita
Plant Pathology
University of California - Davis



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