Some expression strains just don't store well. We used to see this all the time in my lab - a protein will express well at first, then over weeks or months, as you go back to the same freeze down, levels get lower and lower. Toxic proteins (like, say proteases) and leaky promoters (like in pQE vectors) are certainly not a good mix.

What seems to be happening is that the cells will find a way to silence the expression of the gene, and you end up selecting for the cells that express low level or not at all. This happens even at -80. Plasmid extracted from cells that no longer expresses should be fine (it always has been when I have sequenced it again).

The thing to do is simply re-transform your plasmid. You can extract it from the M15 cells, transform a fresh batch and it should work again. Or, ideally, make a storage strain in DH5a or Top10 or similar.

Dear Marisol

I was also doing the same thing in my PhD last year. I was expressing a metallothionin protein in the same system of plasmid and cell strain. Once i expressed it but that time i was not having the proper colums for purification of protein so i just kept the culture in freeze. After 2 months when i started again, i got the same problem. I could not understand that. What i did, i again cloned the gene in fresh plasmid and again i got the expression. I think the pQE series vectors are not for storage. I know it is not a solution but i am sharing my experience, that might help you. If you want to keep that culture, you have to revive plates regularly. One more thing you can do, try with more concentration of IPTG during induction.

All the best Dear Friend.

Regard

Hi Marisol

The problem could be in the storage of your transformed cells. Inappropriate storage can lead to loss of plasmid, as a result of which you are not getting the proteins expressed. Whenever you are sub-culturing, make a glycerol stock and store it in -80 deg C.

If no transformed cells are left, then you transform the cells again and maintain proper stock at appropriate condition. I hope this might help you.

I came across same problem but, it got solved by following the above process.I hope that this might help you.

Regards

Amit

I'll second Amit here. Every time I wanted to express a protein in E. coli or yeast, I transform the cells de novo.

Some expression strains just don't store well. We used to see this all the time in my lab - a protein will express well at first, then over weeks or months, as you go back to the same freeze down, levels get lower and lower. Toxic proteins (like, say proteases) and leaky promoters (like in pQE vectors) are certainly not a good mix.

What seems to be happening is that the cells will find a way to silence the expression of the gene, and you end up selecting for the cells that express low level or not at all. This happens even at -80. Plasmid extracted from cells that no longer expresses should be fine (it always has been when I have sequenced it again).

The thing to do is simply re-transform your plasmid. You can extract it from the M15 cells, transform a fresh batch and it should work again. Or, ideally, make a storage strain in DH5a or Top10 or similar.

What about a mutation in the sequence?

@Laszlo, It is highly unlikely that the plasmid will mutate. There are multiple copies of the plasmid within each cell, and each would need to mutate to silence expression. I have sequenced the gene + promoter region before to check this, and seen no changes. Retransforming into fresh cells regains expression.

My experience is a bit different.

I used to work with a construct and for some reason several mutations occurred in it from time to time. I think it depends on the gene and the host strain.

I agree with you using a single colony after new transformation also helps.

I always checked the presence of the plasmid in the bacteria.

Not only before induction but also before harvesting the cells.

A short comment on copy number.

Multiple copies if you use a high copy number plasmid. usually low copy number plasmids are used. I have to admit I do not know this expression vector.

Protein expression is a pain sometimes, for sure.

pQE vectors are pBR322-derived, so low copy number. But still, that is 15-20 copies per cell. A single bp mutation in one copy in one cell would take many generations to become dominant in the population unless there were extreme selection pressure for it.

But, now that I think of it, I did have one plasmid once that deleted the promoter region during routine growth. That was "homemade" dual promoter plasmid.

So, let me say I strongly doubt plasmid mutation is the cause in this particular case, but it is a possibility that should be checked into if all else fails.

And yes - it can sure be a pain ;-)

thanks so much to all of you for your comments and suggestions. I will transform again and I ll let you know if this was the problem.

Best, Marisol

Hi Marisol. I have just been through lots of trouble shooting dealing with an insoluble protein that would only express when we removed the signal peptide. Best to look in the insoluble pellet with 8M Urea to see if any proetein is here also. Some proteins may only be partially soluble. I think that keeping a stock of DNA to freshly transform your cells is best idea. Also check insert is there by miniprep before using this colony. DNA quality will be poor but it worked OK for me. Grow 1X 10ml culture from single col O/N and dilute1/50 in fresh broth, grow for 3-4 hours then add usually 1mM IPTG 3 hours then pellet. We use BPER from Pierce plus inhibitors and then imidazole purification for native purification or urea for inclusion bodies. This takes lots of trouble shooting to get protein clean and free forom bacterial contaminants, but worth it. We know have rabbit polyclonal antibodies that give excellent response! good luck!

Hi Everyone,

The expression did not work again even after doing transformation from fresh DNA. I tried several times, but unfortunately it was not in the pellet either. I am thinking I may have to remove signal peptide.. any other suggestions??

I removed the signal peptide and it worked for a while, then it started to give more problems again (low expression). I decided to subclone it into pQE30 (without the xa) and it worked. You all are right the pQE systems are very hard to work with, best thing to do is to save lyophilized aliquotes of the DNA and transform every time you start seeing low expression levels. 

I hope this helps everyone.