Hi guys,
I am over-expressing the protein MLKL along with various tagged forms in an MLKL KO cell line. Despite being able to being able to visualize the protein by both immunofluorescence (GFP-tagged version) and through western blotting the protein does not appear to be functional 24 hrs post transfection. My functional assay is induction of necroptosis which leads to the phosphorylation of MLKL and the Phospho-MLKL signature can be detected by western. I have validated my antibody in WT L929 cells and see clear P-MLKL after 2 hrs and significant cell death. However, in my reconstituted MLKL KO cell line I see no such increase in P-MLKL or cell death.
Any help would be appreciated.
Best,
There could be a few different explanations that come to mind. When you add a GFP tag, it could be interfering with the folding or function of the protein. An important thing to take into account is the placement of the GFP at the N- or C-terminus when you have any functional domains near those termini. Another thing is if the phosphorylation site is in proximity to the GFP tag which means the phosphorylation site is physically blocked from being phosphorylated. Some other things to look at is if you have any mutations or deletions in your construct which could be causing problems. If a certain protein is not that well studied with tagged versions, it is best to design and test constructs that have your tags at different ends of the protein. If the GFP tag is causing the problem, you may want to try an alternative tag like HA or FLAG or MYC.
Anthony is very correct. Very often point mutations, especially point nonsense mutations occur during this process resulting in a premature stop codon, or a point-nonsense codon in the transcribed mRNA, and in a truncated, incomplete, and usually nonfunctional protein product. Most significantly are frame-shift mutations, where only the first 28 amino acids might be correct.
may be that depending of the structure of protein and type of charge it on the surface
@ Anthony. Thank you for your response. I feared that the GFP might affect function since the phosphorylation site is near the C-terminus. This is why I designed GFP-MLKL and MLKL-HA constructs and I still do not see phosphorylation occurring. Furthermore, I also engineered an untagged version and even it does not appear to rescue function. I will say that when I was making the construct I had to alter a significant portion of the DNA sequence to reduce complexity and remove certain restriction enzyme sites so that it could be manufactured. While the translated protein should be same as native MLKL the DNA sequence I used vs the cDNA of native MLKL are pretty different.
I see you covered most of your bases. I was going to say that MLKL may also need dimerization or multimerization to function properly as well, but if the untagged form does not work, that raises many other questions. When you sequenced your constructs, did you ensure that both ends are correct? Another thing is to make sure you are expressing the correct transcript variant, or else you could be making a nonfunctional isoform. Now for the modifications, it is hard to say depending on the location of where those modifications were made.
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