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Increase toxic protein expression?

protein expression

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#1 jennifer6271



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Posted 29 August 2011 - 10:36 AM

Hello all,
Here is my situation. I am able to generate my GST-tagged fusion protein only under very specific conditions and at very low levels. After much optimization it turns out that my so-called inducible expression plasmid (pGEX;BL21(DE3)) does not like to be induced when associated with my protein. I have found that I can only get protein expression when I incubate my flask at room temp. for 24 hours at 150 RPM with no IPTG added. With these conditions I can still only get micrograms of protein/liter. Apparently my protein is toxic and the only expression is a result of the leaky promoter. Any suggestions for increasing my protein yield?

#2 pDNA



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Posted 29 August 2011 - 11:22 AM

you can try to add glucose to the medium to lower basal expression levels ...details see [url="http://wolfson.huji.ac.il/expression/procedures/bacterial/Glucose%20supression.pdf"]here. [/url]
in addition you could try to use the strains that carry the pLysS plasmid to further increase leaky expression ...if basal expression is low you can try to grow your cells in buffered medium to higher cell densities and induce your culture with low IPTG concentrations (0.01 M).

It could be that your toxicity is gene dosage dependent ...so switching to a low copy vector is also worth trying. Some people do lower temperature below 20°C ...e.g. 16°C or 4°C ...sometimes this seem to work ...also worth trying.

It would be interesting what is happening to your culture after induction ...do they keep growing or still cease growth or die and lyse? ...have you checked that?

In addition there are also strains available that tolerate toxic proteins to some extent ...go and watch out for C41 and C43 ...details see [url="http://lucigen.com/store/OverExpress-C41-DE3-and-C43-DE3-Competent-Cells/"]here.[/url]
Never worked with those cells but read it somewhere in a paper!

Good luck!

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