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IPTG induction not observable or noticeable


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#1 Ricky

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Posted 03 December 2010 - 12:23 PM

I use pEXP5-NT/TOPO vector and BL21(DE3)star cell, my target gene is a plant gene but synthesized artificially with bacteria preferred codons. I got my purified protein using cobalt column,however,the problem was when I ran the SDS-PAGE with pre-induction and post-induction samples, the bands existed in both samples but there were not any improvements with IPTG induction. The target band in pre-induction sample perhaps due to the leaking expression of T7. Would leaky expression be just as strong as IPTG induction? Or what's going on with my protein?

I sequenced my insert and the nearby plasmid sequence, there are not any mistakes.
Here is my procedure:
Grow in 37C when OD600 reaches 0.6~0.8,
Separate the culture into two halves, one half continue to grow at 37 for 2hrs (OD600 around 1.26), the other half was induced with IPTG final concentration 0.5mM and continue to grow at 37C for 2hrs (OD600 around 0.94). Then harvest cells, sonicate, purify.

In order to improve protein expression, I tried both 25C and 37C, but no differences showing on gel.
I also tried IPTG concentrations range from 10uM to 5mM at 37C, no differences, either.
I also ran the whole cell protein and there were no inclusion body showed up.
Target protein may not be toxic, because I have the same gene in pGS21a vector, and the expression increased a lot with induction by 0.5mM IPTG.

Just to get rid of GST-tag I reclone my gene from pGS21a vector into TOPO vector, but the above problem happened.

Any suggestions would be helpful.

Thanks.

#2 protolder

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Posted 06 December 2010 - 11:46 PM

Hola, this is a normal phenomenom when you donīt use strains pLys or moreover LacIq , wich repress expression untill inducer is added. If your protein isnīt toxic harvest cultures 4 hours after induction and if you want to have much more protein try to use terrific broth TB. Check genotypes of all BL21 strains to understand what each surname means and probably you found the cause of the scape of expression without induction. Buena suerte

#3 Ricky

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Posted 07 December 2010 - 11:30 AM

Thanks.
Leaky expression is all right for me, the main problem is IPTG can not induce more than leaky expression. While pLys strain can repress basal expression, then may see IPTG really works.

Now I worry about whether my protein is toxic or not, I did not see any growth inhibition, but quite not sure whether the cells lyse after induction with IPTG. If it is slightly toxic, then I think pLys strain is quite suitable.

Hola, this is a normal phenomenom when you don´t use strains pLys or moreover LacIq , wich repress expression untill inducer is added. If your protein isn´t toxic harvest cultures 4 hours after induction and if you want to have much more protein try to use terrific broth TB. Check genotypes of all BL21 strains to understand what each surname means and probably you found the cause of the scape of expression without induction. Buena suerte


Edited by Ricky, 07 December 2010 - 11:30 AM.





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