Dear all,
I am trying to purify Citrine tagged protein through agro-infiltration into Nicotiana benthamiana. However, after IP, I always detect a large amount of Citrine but very little or no complete fusion protein. I used very simple protein purification protocol.
After harvesting by liquid N2, grind into powder, add extraction buffer (50 mM Tris-HCl ph 8.0, 150 mM NaCl, 0.2% NP40, with complete protease inhibitor). Sonicate 3 times with 15 second/1minute break in ice water (with bioruptor), leave on ice for 30 mins for extraction. Centrifugation 4 degree full speed two times to remove all particles. Add IP beads to supernatant 4 degree 1 hour, spin down and wash 2 times with extraction buffer, then elute by boiling with 50 ul 2x Sample buffer. Subject the elutes to SDS-PAGE and WB.
Does anyone have the same problem before and can help me to solve the problem? Thank you very much!!
Cheers,
Kuan-Ju Lu
Please post a gel image. Here is what you need to do in order to help work out what is happening and for us to help:
1. Run the lysate BEFORE IP on a gel and western blot - are you sure you have lots of the fusion protein present in the sample to begin with? Is there both citrine and fusion protein? You need to check to see if the protein is intact prior to IP before you can assume the problem is in the IP stage.
2. How are beads prepared? Do you pre-conjugate them with the anti-citrine antibody? or is the antibody directed against the fusion protein?
Please post images of the gels so I can be of more assistance.
Hi Kuan, proteins don't usually break. They are mostly hydrolyzed into smaller peptides by proteinases.
If you included a complete proteinase inhibitor cocktail with EDTA then the only way I can see that you can get protein fragmentation is through excessive sonication.
Hi Kuan,
it is not uncommon to see proteins being "degraded" in plant transient gene expression assays. This is because the plant cell has many proteases and the Agro-infiltration is inducing stress responses as well. We have seen fusion proteins preferably being cut within the junction (linker) between the two proteins, this is because this region is unfolded and often accessible, thus prone to proteolysis. In my experience, if this happens it happens already during the incubation phase, i.e. you cant prevent it by adding protease inhibitors. Here are some suggestions how to circumvent / solve this problem:
(1) ensure your plants are vital and are exposed to as little stress as possible (it all keeps adding up).
(2) try to target your fusion protein to different cellular compartments.
(3) If you have purified protein (and degradation products) send them for N-terminal sequencing to determine the cleavage site and consider changing the sequence in this region.
(4) Sometimes a time-course analysis is helpful to understand when the degradation occurs, i.e. harvesting earlier may give you a higher percentage of intact product.
(5) Asp-Pro is a labile peptide bond, i.e. you may want to check your sequence for that.
(6) in cases where proteolysis indeed occurs during / after extraction you may want to look into different buffers (pH) or heat treatment.
See e.g.
Article Heat-precipitation allows the efficient purification of a fu...
Article Optimized Blanching Reduces the Host Cell Protein Content an...
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