I have a clone of a protein in pET21a and have tried expressing it in BL21DE3 cells and recently BL21DE3 star .On both occasions I have found loss of expression. I see a protein band with freshly transformed DNA but once I scale up from glycerol stock I do not see the same level of expression.
What are the reasons this happens? Is it because of the unique way in which the plasmid enters the competent cells?
Hi there,
BL21(DE3) transformed cells are not stable then you can't rely on a frozen stock for conservation. An othe rissue is that from one transformed cell to an other you may observe very different level of protein expression. So what I usually do is always start expression experiment from a fresh transformation mix which I culture overnight (ie not starting from an isolated clone) and use for inoculation. By using a mix of transformants it is averaging the effect of clone to clone variation in protein expression level.
Hi Dominique
Thanks for yr answer .Another approach could be to take many colonies after transformation -same principle I guess .Please see this paper I read it seems to indicate that fresh transformation is the only solution.Im a bit surprised that such a commonly occurring problem has not warranted more serious research and investigation but here is 1 paper that addresses it.Trust me these 11 months have been horrendous for me as I kept making glycerol stocks every 3 months thinking everything will be fine but --- kapot---that explains it.Thanks again
What kind of protein are you expressing? Is it an E. coli protein?
In principle what can happen is that your plasmid might undergo homologous recombination into the geneome. That is - if a part of your construct is homologous to a part of the genomic sequence there is a selective pressure in a culture to favor the cells that have recombined the plasmid DNA into the genome - they no longer produce the protein, since a part of its sequence is missing, but are still resistant to the selection marker. This behavior (at least presumably) was observed in L. lactis while overexpressing a L. lactis protein - after some time you have resistant cells that do not express the protein from a plasmid. In theory cells have the necessary enzymatic machinery to facilitate the process as a part of DNA repair system.
I m expressing a mycobacterial protein it s size is 18Kda .The protein is functionally a chaperone heat shock protein
If I were you I would look into homology of the protein to E. coli proteins. All that is needed for homologous recombination is sequence identity. If you look into that and find no nucleotide sequence homology then you might be quite certain that it is not the issue. In the L. lactis case I mentioned above the issue was fixed by knocking out the genomic sequence, so that the one on the plasmid is the only one present in a cell.
Did you check if the protein has negative impact on the cells? If you overexpress a chaperone from a different organism, I would imagine it might keep a certain protein/proteins unfolded/bound in a cell, rendering it less viable. That would also account for a selection towards cells with lower protein expression levels, but still resistant to the selection marker. It is a long shot though, I have no particular example to back up this idea.
I will need to study up all you mentioned.By the way there is a protein in E.coli called SlyD that is also a chaperone and acts on the same substrate as my protein.When I made soluble protein this protein interferes by binding and eluting form the Nickel NTA column(my protein has his tag).Analysis by mass has confirmed SlyD in my preparation.
The issue is I dont know if the chaperone activity im getting is due to my protein or the background proteins like the One mentioned above.
Thanks for yr help.Will need to read up on this to unds your hypothesis
Hi Gautam,
Regarding your issue with SlyD contamination in Nickel NTA purification, here's a recent article with an example of SlyD contaminant removal by affinity chromatography. If your target protein eluted along with SlyD as the only contaminant, this may help:
http://www.sciencedirect.com/science/article/pii/S1046592817301651
Hope this helps,
Eric
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