Recently I have big problems to express my recombinant protein although it worked quite nice some months ago. It is an Arabidopsis protein cloned into pET22b. I'm using Rosetta2 (DE3) cells for expression because there are some rare codons.
My workflow is as follows: I'm preparing overnight cultures of 5ml LB and inoculate them with single colonies from freshly transformed plates. The next day I incoculate bigger cultures (50-500ml), usually in 1:100 dilution. My problem is that in these bigger cultures the cells start to behave strange: First, they need a long time to reach OD 0.6 at 37°C (around 5h or more; when I did this before it took a maximum of 4h). Then, when I induce the expression (IPTG 0.25mM - 1mM), I let the cells grow at 37°C (I also tried 30°C) and harvest after 3h. Before this was the right timepoint, there was nice expression and the cells continued growing. After 3h they reached an OD of ~1.2 - 1.5. Now it seems they stop growing, so 3h after induction the OD ist still 0.6. When I do SDS-PAGE I can see that the expression did not work.
Even stranger: I tried to set up small cultures (2ml) from single colonies, let them grow to OD 0.6-1 (obviously in plastic tubes they easily reach these higher ODs) and induced the expression: Here it worked reproducibly (they continued to grow, so that they reached OD 1.5 in the end) and in the SDS-PAGE I could see my protein already in total cell extract. But when I inoculated a big culture with the same cells (of course before induction) the same problem appeared.
So I thought there might be a problem with the glassware, and I cleaned the flasks very well with EDTA-solution and acetone, but the problem remained the same.
Does anyone have an idea what could be wrong?
Hi Daniel,
since you already got some protein out of your cells with this exact protocol, it is indeed very strange.
A few points to consider: Upscale volumes not always have a propotinal increased yield. Try using a bigger flask for your culture to get a better surface:volume ratio and shake for at least 180 rpm.
If you see that your colonies grow at a slower rate inoculate with a lower dilution (e.g. 1:50 - 1:20). Also keep a small scale tube as a control to see if the cell grow is batch dependent low.
Worst case you can grow in multiple 50mL falcons to get enough volume for your purification (but it wouldn't solve the strange behaviour).
Do you think that your protein is toxic? If expression is leaking before induction this can inhibit bacterial growth-add 1% glucose final to all your plates and media (50% stock is convenient to make up).
If after slow growth they suddenly take off and then crash, followed by re-growth over an extended time period you could have a phage contamination that is more likely to occur at large scale than small scale. If you finally get any biomass from infected cells it will almost certainly not contain your protein.
This sounds like a T1 phage infection to me, as it only happens in the large scale cultures. Bleach all of your flasks and bake them, and make new competent cells from an uninfected glycerol stock. Also, the incubator for the large scale cultures should be cleaned. Hope this helps.
Thanks for your suggestions. I already tried many things such as inoculation with a lower dilution (didn't change anything), and using bigger flasks. I'm also quite sure that the protein is not toxic, as it already used to work. I'm afraid that it could really be a T1 phage infection. How could I test this? And why do I see the effects only in big cultures?
First nobody asked (including me) what volumes you are using in your flasks? To retain good aeration levels 700ml is the absolute limit in a 2l non-baffled flask shaken at 225 rpm. If you haven't exceeded that 30% fill volume in your flasks and you don't have too much foaming then the problem is probably not aeration.
If they are contaminated with phage before/during transformation you may see odd-shaped bacterial colonies, star-shaped or like a biscuit with bites taken from the edges. Do you have similar problems expressing other proteins or just this one? Anyone else in the lab having similar problems with reproducibilty?
If you see this you may have to switch all your bacterial strains in the lab to phage resistant. If you cannot buy resistant strains and you suspect phage you can normally derive your own very easily by taking a centrifuged, 0.2µm filtered supernatant from a phage lysed culture and co-plating say 100µl of this with an aliquot of fresh cells (aliquots that you would use for transformations are fine) on LB Agar plates plus any antibiotic that the strain itself carries. Do a control with cells only and you should see a dramatic difference in the number of colonies-a lawn probably in the absence of the phage and perhaps tens of colonies on the plates with phage.
Pick individual phage resistant colonies. Make sure they grow normally without lysis etc., prep glycerol stocks and competents from them as you would with normal cells. Make sure that they behave normally for cloning or expression of a control protein depending on the strain. A couple of weeks work but if you have phage problems they will just keep on holding you up in a really frustratingly random fashion for ever.
As an update: I tried different things in the meantime and now it's working perfectly again. I changed the following: First, I further decreased the volume in the flask (e.g. 200ml in a 1L flask), although I don't think that was the main point. Additionally, and I think that's what brought the sucess, I added 1% glucose to the liquid media, as suggested by Nick. Now the cells grow happily again and the expression rates are amazing. So maybe the expression was really leaking, although it's strange that this appeared from one day to the other. Anyway, thanks for all the suggestions.
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