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Expression in codon plus of GST fusion protein - (Dec/16/2006 )

friends,

I am trying to obtain a GST fusion protein in Codon Plus (RIPL Stratagene) because I have problems of Use codons for my protein (rare codons for Arg in e coli)

I am using the vector pGEX-2TK, aplying induction for 5 hrs with 1mM IPTG.

After the supernatant is mix with Sepharose GST and on gel PAGE, I only see a Duplet in 25KDa, which can be only GST...my protein have 7KDa then the fusion protein should be of 35KDa, but I do not see this protein...

The same result I see on normal BL21, then Codon Plus are not help me!!!

I send the PCR product with primer pGEX forward and reverse of my clon and all is in frame....

Will need others conditions for express my fusion protein?
Will be toxic my protein? Will be in the pellet?

My protein is very hydrophobic, but I hope that with GST will be in the supernatant...

How is possible that express until GST and drop, if the codon plus have the plasmids for tRNA rare in e. coli??

please help me!!!!
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-javo-

you mentionned your protein is very hydrophobic. So i would think it goes to precipitate.
Did you try commassie to see if your protein is at least expressed ?
second : may you play with UREA during your purif to reduce precipitation?

-fred_33-

QUOTE (fred_33 @ Dec 19 2006, 08:38 AM)
you mentionned your protein is very hydrophobic. So i would think it goes to precipitate.
Did you try commassie to see if your protein is at least expressed ?
second : may you play with UREA during your purif to reduce precipitation?



When you say "precipitate" is that the fusion protein is in pellet?

I have GST in N-terminal of my protein fusion and when express the fusion protein only express GST (i see this in PAGE and commassie blue R-250 stain) because when mix with sepharose GST (purification) only appear in gel a protein of 25KDa... the fusion protein is 34KDa...


the sepharose GST (used for purify) can bind my fusion protein when urea is present??

thanks!!!

-javo-

i mean the protein precipitates and thus acquire the nighmaring capacity to go in the pellet during centrifugations (so in the elution centrif too).

Maybe sounds reared to you but did you try to load a few portion of the beads (just add 10┬Ál Laemmli 5X n your beads denature and load all the mix) to check for presence of your protein in that pellet?

even with your reply i did not understand : do you see the 34kD form?

you need to do some biblio to check for capacity to bind in urea supplemented conditions (or ask the technical service of the comany where you purchased the GSTbeads
But i would give it a try.

-fred_33-