hello all
I am expressing a His6x recombinant protein and after lysis in lysis buffer i sonicate the cell suspension (40% amplitude 30 sec ON 30 sec OFF , 5 cycles). when i run SDS PAGE of the pellet and supernatant from the above step. i am able to see specific band of my recombinant protein in both pellet as well as supernatant . I am not sure how to proceed further for purification. Please guide me in this regard
As Roger said, not unusual. There are several possible reasons:
- Already during your production, you protein is not able to properly fold quantitatively, and distributes between inclusion bodies and a properly folded, soluble state already in the cell - In this case, you might be able to push your system towards more completely soluble expression by expressing at lower temperature, while changes in the sonication protocol will have no effect.
- Incomplete disruption of your cells: let your sample cool down, then do another round of sonication and see whether the ratio between the amount of your protein in the supernatant and the pellet improves.
- Denaturation of the protein during sonication - In this case, multiple rounds of sonication shift the distribution between supernatant and pellet towards the pellet, indicating that you need to change to a gentler protocol.
Hi Ankit,
This situation is relatively normal, especially for the target protein whose properties are not stable enough. It is recommended to obtain soluble protein of interest by purifying a large number of cultures, paying attention to keeping fast, and the temperature low during the purification process.
Good luck!
As Roger said, not unusual. There are several possible reasons:
- Already during your production, you protein is not able to properly fold quantitatively, and distributes between inclusion bodies and a properly folded, soluble state already in the cell - In this case, you might be able to push your system towards more completely soluble expression by expressing at lower temperature, while changes in the sonication protocol will have no effect.
- Incomplete disruption of your cells: let your sample cool down, then do another round of sonication and see whether the ratio between the amount of your protein in the supernatant and the pellet improves.
- Denaturation of the protein during sonication - In this case, multiple rounds of sonication shift the distribution between supernatant and pellet towards the pellet, indicating that you need to change to a gentler protocol.
Finding a recombinant protein both in the pellet and in the soluble fraction is the norm, rather than an exception. Solubility is not an on/off property. If the amount in your supernatant is enough for your experiment, just go ahead and purify the protein from the supernatant, ignoring the insoluble fraction. Otherwise, you may play a bit with the growth and purification conditions to improve your yield of soluble protein, as other have suggested. For example you can lower the temperature of growth of the bacteria and extend correspondingly the induction time - this often it improves the yield.
Hi Ankit,
I agree with all the above responses and you may try to use 'Inclusion Body Solubilization Reagent' from thermofisher to solubilize your protein from pellet if most of your protein is there and you could NOT solubilize it with temperature and lysine optimizations.
Regards
Isiaka
Use modified purification protocol under denaturing conditions as recommended in manufacturing protocol. To use the protein care must be taken, as protein may loss of its correct 3D structure due to breaking non-covalent bonds- lose the proper folding !
In my experience cell disruption using sonication is rarely 100%. Because of that, when working with a new protein, I like to include a low rpm centrifugation step to remove all the remaining cells from the lysed material. Then I centrifuge with high force and analyze the pellet and the soluble fraction. From that you should be able to determine how much of your protein ends up in the soluble fraction, and how much in inclusion bodies.
Good luck!
Ankit Kumar, you've already got high-quality answers from the experts. I want to add that I faced a similar situation with my protein as well- where the protein was fractionating in both periplasmic and cytoplasmic space when tested with isolation buffers of both. I just pooled both the fractions, and it gave me a higher yield than both of them individually. A wise approach would be to test your protocol as stated by Annemarie Honegger , optimization with lower temperature, lower RPM and very importantly changing your strain (as Lemo-21 and C-43 worked for me instead of conventional BL-21 - expression shifted from inclusion bodies to cytoplasm and periplasm with the help of a suitable tag). All the best.
Aditya Trivedi , protein that has been exported to the periplasm (and may form inlusion bodies there) can usually be distinguished from protein that remained in the cytoplasm by the absence (periplasm) or presence (cytoplasm) of the signal peptide
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