hi to all,
I'm trying to express a protein in E.coli bl21DE3 gold. this protein is about 70 aa long and is very similar to a metallothionein, having 6 cysteines. the plasmid is optimized for the expression in e.coli.
I tried several expression conditions in order to express it:
-inducer concentration;
-temperature;
-time;
-aerobic and anaerobic conditions;
unfortunately the protein was not expressed in none of that conditions. i checked it by means of sds page, nmr and i tried a purification/lc-ms. but nothing.
what does it mean?
is it possible that the protein is digested by the proteases when it is expressed? can you provide me some literature about similar problems?
if i try to induce the protein in the presence of metal ions in aerobic or anaerobic conditions, should the situation change? why?
Hi Antonio,
I encountered a few years ago a similar problem with another protein.
May I ask you first what is the transcriptional control,of your recombinant coding sequence? This could indicate us a way to escape the nonexpression deadend.
Best regards,
Philippe
I would first double check that the plasmid is ok. Which vector are you using? Check if everything is there: promotor, RBS, Start and stop codon (in frame).
The cytoplasm of E. coli isn't conducive the formation of disulphide bond formation. It could be that your protein is misfolding and aggregating as a result. Likely this would direct it to the proteolytic degradation machinery. Have you tried putting a periplasmic signal sequence on the protein? Or expressing it in an E. coli cell line which has disulphide chaperone activity/reducing conditions knocked in (Eg. Shuffle T7)?
Make sure you have a positive control (protein purified from another source or commercially produced) to make sure your detection assays are working the way you expect. In certain cases, detecting small MW proteins is not trivial, so false negatives could be a concern. In E. coli, it is certainly possible your protein is either secreted or degraded. You might have better success fusing it to a larger protein (GST, MBD, KSI) or by targeting it to the periplasm.
Your sequence might not be optimal for efficient translation in e.coli. I agree that fusing your sequence to a well behaving protein such as Thioredoxin, MBP, GST or SUMO might dramatically improve expression. I normally systematically screen for a number of tags and observe regularly an improvement with the latter. However for small proteins it would be advisable to try the smallest tag available. In that regard, thioredoxin (17 kDa) might be a good option. It might also help in the proper formation of disulfide bonds if you express it into cells with a more oxidative cytoplasm such as Origami or Shuffle
Hi Antonio,
I think your protein is not secreted nor degraded but rather is misfolding
Best wishes,
Ilya
hi to all, thank for your answers!,
at the moment i think i will try to express the protein in lemo21 e.coli. i read that the protein in this strain is translocated to the membrane, and this strain finctions as a plysS.
what do you think?
Hi Antonio,
Lemo21 are basically BL21 cells, just that you can regulate the T7 lysozyme expression. PlyS and Lemo21 are made to reduce the expression level.
I am afraid that won't improve your situation. What kind of plasmid are you using? Do you have a tag on your protein? any possibility to do a Western blot?
hi tobias, i'm currently using pet21 plasmids. i have tried to express the protein with and w/o the his tag, but no expression was detected, also by means of western
so you are sure that your plasmid is ok?
It seems you might need to consider changing the plasmid according to the suggestions above. You could also try to express it into the periplasm (using pet22b), that might help if your protein gets degraded in the cytoplasm.
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