I have cloned a mammalian gene both into pet 28a and pgex-4t2. My protein is expressed properly in BL21 containing pgex-4t2 (I have checked the functional activity of my protein). However, cloning into pet28a is seeming to give some toxicity- reduced cell growth and viability. Can anyone explain this behavior and exactly how should I approach the problem now? The reason why I want to go for pet28a is that my protein is very difficult to solubilize when tagged with gst in pgex-4t2.
Some recombinant proteins are very toxic to E.coli. One way to solve it, is to use a bacteria strain suitable to express toxic proteins. In the past I used BL21-AI with excellent results. You will also need arabinose to induce the expression and it works with plasmid with a T7 promoter
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@Jorg
My protein is 38kDa. It is getting expressed properly with GST tag in pGEX-4t2. The toxicity problem is coming when cloned into in pET28a.
@Oscar
I have tried BL21 pLysS. This has yielded similar results,ie, toxicity problem.
Thank you... how can I figure out exactly what is causing the toxicity in pET28a? Because my protein with GST tag works fine...
Dear Archita, This problem is associated with an expression host what you are using not because of the vector system so you can make a trail with the new generation expression system. There are numerous expression host system commercially available for expression of mammalian genes with high expression levels which difficult to get in conventional vectors. Archita, here I suggest two host to have E Coli Rosseta or BL C41, C43 (DE3) by using same vector.
What do you mean by toxicity in your system. You can check the medium concentration you are using.
Try Studier auto-induction media. There is no problem with the pET28a vector as such. Every vector just needs some adjustments for the expression. Studier auto-induction media works much better than any other media for pET28a. Good luck.
Dear Archita, pET28a and pGEX use different promotors. pET28a usally give higher expression level under the control of T7 promotor. In my opinion, this high level foreign protein expression level lead to its toxity. What you can do is to slow down its expression such as lower growth temperature or low IPTG concentration.
Thank u...i am now comprehending the problem.. I have tried lowering the temperature to slow its growth but the problem persists...
I think trying different systems as suggested would be a better idea... For now i am sticking to gst as it is giving protein expression... Thanx a lot...
Archita ,
Have you tried adding 2% Glucose to your medium (including plates)?
You use LB, right?
I found adding Glucose quite effective in suppressing toxicity from mammal genes in pET vectors.
Also, please check your plasmid for mutations when the insert is toxic. The least toxic random mutation can accumulate in E.coli pretty fast.
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