I have cloned a cyanobacterial Aldo-Keto Reductase gene in pET/BL21 system and tried to express protein at lower temperature (16c) and IPTG concentration (0.05mM) to avoid inclusion body formation but still I am getting my protein in inclusion bodies. Does anyone have any experience with pCold vectors (Takara) or is there any other better remedy of this problem?
Hi Chhavi,
Have you tried to express your protein in the presence of Alcohol(Ethanol).
Grow your culture in the presence of ethanol at different % levels (1, 2, 3, 4,5) Maximum 5. at 37 degrees temperature up to OD reaches 0.5 and later induce the protein and grow at low temperature (30, 23) for over night.
Try this procedure. All the best .
Ramesh's is a good tip, and it does not take a lot of work to try it.
The bad thing about the pET/BL21 system is that it takes over the cell upon induction; so most of the target protein is actually produced during a relatively short period of time (This is aggravated in autoinduction media, although that is not your case). Some proteins are just not born for this and will aggregate.
pCold may definitely help, as probably will other -weaker- promoters such as straight plac, which will enable you to produce the target protein at lower levels, but accross a longer time.
You could also try the chaperone kit from Takara.
You may try different expression strains. We had success with some proteins by changing from BL21(DE3) to JM109(DE3) - available from Promega (although not as competent cells, you'll have to prepare the competent cells yourself). It seems to grow slower and give our proteins more time to fold.
Btw, make sure to always call the cells BL21(DE3), because the DE3 lysogen expressing the T7 RNA polymerase is essential. I know about two important delays caused by people thinking BL21 is the same as BL21(DE3) and JM109 is the same as JM109(DE3).
Thanks Ramesh. i will give it a try.
Thanks for mentioning JM109(DE3) Mark and also for correcting me. Yup its BL21(DE3).
Thank you Pulicherla for sharing your experience with pCold. Please let me know if something works.
Dear Chhavi,
I had remarkable and reproducible expression of soluble recombinant fusion protein using the system pCold TF DNA on BL21(DE3) E. coli. Parameters of the cultivation: 15°C / 36-40 hours / medium volume:flask volume < 1:5 / 0,4 mM IPTG @ OD600 = 0,6-0,8. TF tag helps to keep your protein soluble, but then you have to optimise the cleavage of the tag, and purify your target (unless you can do your investigations on the whole fusion protein). There are three cleavage sites for different proteases; in my hands thrombin worked really good, but if you have also the others, just test them. Good luck!
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