How to improve IPTG induction? - (Mar/14/2007 )

I am trying to express and purify a GST-fusion protein now, but the IPTG induction effect is not satisfactory... There is no big different between uninduced and induced groups...

I have two concerns:

1> The target DNA was ligated into pGEX vector, then I transformed it into BL21 (DE3) Star competent cells from invitrogen, is this okay since that pGEX vector is not T7 promoter driven but BL21 (DE3) Star cells contain T7 RNA polymerase.

2> When I used IPTG for induction, I used 1 mM IPTG as the final concentration and incubated cells at 37oC for 2.5 h with a OD600 above 2, is this okay??

Thank you very much!!

-Jason

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Do you mean you start inducing at an OD of >2? If so, that's way too high. You need to have your cells in log phase, and at an OD of 2 your cells will be in vegetative phase, which means very little protein synthesis. Try inducing at an OD of 0.7 to 0.8, and you should find much better yields. Your cells seem OK, but you should also be able to induce with 0.5 mM IPTG.

Happy expressing!

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sorry, maybe i didn't say it clearly, i started to induce when OD600 is around 0.6-0.7. The OD600 was above 2.0 after 2.5 h incubation.

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Have you grown your cells with appropriate antibiotics? Because if not, maybe the cells you get are not the one having your plasmid.

Also, 2.5 hres of induction seems not a lot to me. Maybe you should induce for a longer period. We usually do 5hres.

Hope this helps!

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I grow a starter culture to about 0.6 and then use this to inoculate fresh media 1/100 dilution and then grow this until 0.4 and THEN induce and have had no problems.

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Hi!
I'm also using the BL21 cells and are following a protocol from the company who sold me the cells (Stratagene).
Grow cells in 1 ml media over night and the next morning pipet 50 microliter into 1 ml fresh media (without antibiotics) and incubate for 2 hours. Then induce with IPTG (final conc of 1mM) and harvest cells after 2 hours. It could be important not to use antibiotics because the bacteria use a lot of energy to overcome this pressure.
// Lene

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Hmm not using antibiotics? I understand that bacteria does uses alot of energy for the antibiotics but I think it is very important as well. As long as the plasmid has the antibiotics resistance gene, it will grow.


lenenoerby,

Try to play around with the time expression parameters. I did 2 to 12 hours before and found out up to 10 hours, the protein has the highest level of expression. Why not just try out 5-6 hours (standard time expression). Good luck!

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Hi again!
I know it is very important to maintain the plasmids with the antibiotics - but this is an answar from the company who sells the BL21 cells:
Thanks a lot for your inquiry about our BL21 cells.

We at Stratagene have noticed that the presence of an antibiotic could be
inhibitory to protein expression, and we do not recommend any antibiotic to
be present during induction/expression with BL21 cells. Fighting against the
antibiotic seems to be very energy-consuming for the cell, which leads to
decreased levels of protein expression.

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Hi Jason,


R0 If you dont see a "big difference", maybe you arent producing at all. Make sure that you are producing something.

R1 If you have regular cells like XL1-Blue, you can use them to see if it express better than in BL21. I dont know if BL21 star interferes or not but its always good to check the production in more than one cell type.

R2 In most cases I found than OD 1.0 with 1mM IPTG for 2h at 20ºC gives better results for soluble protein.

I´m also having trouble to produce one protein, and I´m waiting for the sequence to double check it before I continue. I can produce a shorter construct of the same gene but its insoluble ;-)

Cheers

Tiago

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I havent try to express without adding antibiotic. I didn't read anything from Qiagen handbook regarding about antibiotics. They insisted me to add antibiotics for selection.
I still got my protein to be expressed with the presence of antibiotics.

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