In our lab we use C43(DE3) cells to express and purify recombinant histidine tagged proteins. We work with archaeal proteins. I always freshly transform the E. coli cells and proceed thereafter. I used to get decent amount of protein (1-2 mg/ml). Recently I am having trouble getting enough protein. It seems that there is something wrong with induction. My protein of interest is not getting expressed all of a sudden and I am getting very low amount of protein in the elution (200 ug/ml). The SDS-PAGE also revealed faint bands in the elution. I have tried various things for troubleshooting purposes.
1. I have changed my clone and started from the initial glycerol stock.
2. Changed the concentration and stock of IPTG
3. Changed the culture flask volume to increase aeration.
4. Varied the induction temperature 25, 28, 37° C.
5. Tried using very rich media (terrific broth)
6. Tried adding ethanol to the media to increase yield.
7. Tried different strains (Bl21, C43, rosetta)
8. Tried adjusting the pH of the media since acetate production inhibits cell growth
None of these worked.
It would be very helpful if someone can shed some light on what is wrong here.
Thanks in advance.
Hello! Have you checked the expression within whole cell lysates (before loading onto the column) and the column flow through? Given that you've checked numerous variables on the expression side, it could be that you're expressing the protein, but aren't seeing it in the elutions because it can't bind to the column and is coming out in the flow through.
Hello Michael. Thank you for your valuable insights.
Yes I have checked the whole cell lysate and flow through. The expression of my protein in the whole cell lysate is very low. But, I have observed that this low amount of protein is binding to the column since there is nothing in the flow through. I have analyzed each fraction in SDS-PAGE. Also, keeping this in mind I tried recharging my Ni-NTA column, but without any significant increase in the amount of protein.
Hi Koustav,
I see that you've gone back to the inital stock and tried different cells lines, but have you tried re-transfecting a stock BL21, etc line or remaking the stock IPTG?
Thank you,
Michael Jaskolka
Yes, Michael. Whenever I have to purify my proteins of interest, I always start with freshly transforming my cells. Here also, I tried transforming stock BL21 with the clone. Regarding IPTG, I made fresh stocks and also considered increasing the IPTG concentration but without any luck.
Hi Koustav Bhakta
I recommend the following reading, as it suggests why low protein overexpression can occur or can be inconsistent between batch cultures.
Article Protein over-expression in Escherichia coli triggers adaptat...
I experienced this myself and was able to remedy. You can do a simple experiment by plating your culture after induction on media with and without your expression plasmid's selective antibiotic. I noticed immediately that my protein was highly toxic and most of my cells during my "standard" expression were in fact no longer carrying the expression plasmid, resulting in inconsistent and very low yields. I also switched from ampicillin selection to kanamycin selection, which helped tremendously - b-lactamase is a very poor selective marker because in batch culture, the extracellular b-lactamase can accumulate, allowing non-resistant cells to grow as early as mid-exponential phase.This was a huge problem in my overnight expressions, resulting in less than 1% of my cell pellet actually containing expression vector and hence almost no recombinant protein.
I suggest screening IPTG concentrations as the above citation does, and then plate cultures and screen colonies from those plates for their expression levels. Streak a colony with high expression for purity and keep a glycerol stock of this for future expressions. By switching from an Amp->Kan vector and doing this, my expressions improved from no detectable protein by Western blot to consistent yields.
Hope this helps!
ACA
Hi Alexander
Thank you so much for the article and the insightful suggestion. I think this might be the case for my expression vector as well. My gene is cloned in pEtDuet vector which has an amplicillin selection marker. So, cells becoming resistant might actually be a possibility. I will carry out the things you suggested and check for the expression of my protein.
Hi Koustav
If your proteins are toxic to the cells, you could try using BL21(DE3)pLysS cells. This strain carries the gene encoding T7 lysozyme. T7 lysozyme lowers the background expression level of target genes under the control of the T7 promoter but does not interfere with the level of expression achieved following induction by IPTG. It's also resistant to chloramphenicol. So you can select colonies on chloramphenicol + antibiotic (plasmid selection marker) plates.
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