4 replies to this topic

[TomE]

Hi all,

I'm trying to express a 180kDa GST fusion protein from BL21 PlysS using the pGex-4T-3 expression vector. I'm confident the DNA is correct. So far I've tried inducing log phase cells for 2hrs. @37C and 4hrs. @30C but neither show the expressed protein by SDS-PAGE. Western blotting with the specific antibody perhaps shows a faint wiff of it in the overexposed films but difficult to say. I've used this expression system before for smaller proteins with no problems. Going to try lower temperatures (21 and 18C) O/N next. Any other suggestions? Also, should I be adding glucose to the LB? I know some do but it hasn't been an issue for me in the past.

Thanks

[protolder]

TomE, on 12 July 2011 - 03:19 PM, said:

Hi all,

I'm trying to express a 180kDa GST fusion protein from BL21 PlysS using the pGex-4T-3 expression vector. I'm confident the DNA is correct. So far I've tried inducing log phase cells for 2hrs. @37C and 4hrs. @30C but neither show the expressed protein by SDS-PAGE. Western blotting with the specific antibody perhaps shows a faint wiff of it in the overexposed films but difficult to say. I've used this expression system before for smaller proteins with no problems. Going to try lower temperatures (21 and 18C) O/N next. Any other suggestions? Also, should I be adding glucose to the LB? I know some do but it hasn't been an issue for me in the past.

Thanks

Hola, if the system has runed before with other proteins the problem , to me, could be this new one, but the fact of a very little band in WB could open a hope window. So if you have home made competents be sure that Cm has been added in the culture for stock preparation .And  add Cm too in the ON plate to seed or preinoculum in order to mantain pLysS. About glucose, sometimes is added  to mantain repression before induction, but your plasmid has lacI repressor too, so in your case I will add 0.5g/l of lactose (natural inducer) at 0.5 absorbance units I will low temperature at 30 or less, and I will follow growth and expression for 4-6 hours or more. Buena suerte

[qzlabs]

protolder, on 12 July 2011 - 09:55 PM, said:

TomE, on 12 July 2011 - 03:19 PM, said:

Hi all,

I'm trying to express a 180kDa GST fusion protein from BL21 PlysS using the pGex-4T-3 expression vector. I'm confident the DNA is correct. So far I've tried inducing log phase cells for 2hrs. @37C and 4hrs. @30C but neither show the expressed protein by SDS-PAGE. Western blotting with the specific antibody perhaps shows a faint wiff of it in the overexposed films but difficult to say. I've used this expression system before for smaller proteins with no problems. Going to try lower temperatures (21 and 18C) O/N next. Any other suggestions? Also, should I be adding glucose to the LB? I know some do but it hasn't been an issue for me in the past.

Thanks

Hola, if the system has runed before with other proteins the problem , to me, could be this new one, but the fact of a very little band in WB could open a hope window. So if you have home made competents be sure that Cm has been added in the culture for stock preparation .And  add Cm too in the ON plate to seed or preinoculum in order to mantain pLysS. About glucose, sometimes is added  to mantain repression before induction, but your plasmid has lacI repressor too, so in your case I will add 0.5g/l of lactose (natural inducer) at 0.5 absorbance units I will low temperature at 30 or less, and I will follow growth and expression for 4-6 hours or more. Buena suerte

PlysS should not affect the expression since it is a tac promoter not a T7. How to start your big culture may play a big role here, might want to try to start the induction earlier than you did before.

[TomE]

So I've tried lower temperatures (30, 24, and 18C) and inducing in Lag phase and I still get nothing by SDS-PAGE and it doesn't seem as though the westerns are getting any better either? Any other suggestions? As far as other cell lines, I know I have DE3. Are there any in particular you would recommend for such a large protein? Thanks for the input.

[protolder]

TomE, on 18 July 2011 - 02:24 PM, said:

So I've tried lower temperatures (30, 24, and 18C) and inducing in Lag phase and I still get nothing by SDS-PAGE and it doesn't seem as though the westerns are getting any better either? Any other suggestions? As far as other cell lines, I know I have DE3. Are there any in particular you would recommend for such a large protein? Thanks for the input.

Hola, I have been checking the lastest issues for protein expression and I have seen two new strains from NENbiolabs called lemo21 and shuffle. See the charcteristics and aplications, but I think that you had to have succes with your done  experiments. Buena suerte