Is the selection pressure oK?  Or may the culture has been polluted with other clone, and the target BL21 is uncompetitive. Be carefule when operate.

Curious case!! Selection pressure not being correct is a very plausible explanation. What is the size of your protein and how much protein do you load? I mean, if the size is small and when you load different amounts of protein of uninduced and induced cultures sometimes you miss the band either due to diffusion (small protein) or sometimes you arent able to make out the band because in uninduced sample the leaky expression also gives a band and If you have loaded more protein you wont be able to make out your induced band (Telling that from experience!!).

You can also try inducing at a higher OD, say at OD=0.8. I used to induce at low OD and used to get low amounts of protein.

Do you think it could be toxic to the cells? Although this doesn't really explain why you see something at small scale-if you think it could be toxic try these:

If you are using the T7 system with IPTG you can add 1% glucose throughout (LB agar for transformations and all subsequent media) as Yoram has already  suggested. This will effectively block expression from the T7 system especially if you use something like LysS/E strains until the point of induction-it will at least allow you to get some biomass before the expressed toxic protein begins to kill your cells.

Auto-induction may also help as the protein is not expressed until the cells are at, or near, saturation so they are no longer in growth phase when induced (the phase where classically you would add IPTG) .

Also you don't say if your cells are growing normally -do you see anything strange in the growth especially a prolonged lag phase or a crash during late exponential stage then re-growth? This could indicate phage contamination that you may possibly be avoiding at small scale by using disposable plastics etc. but not at the large scale with re-used flasks etc.

thank you for your responses!!

so i am adding antibiotic to the culture to keep the selective pressure. i have been inducing around 0.5 OD but I have also tried doing it at 0.6 or 0.7 and it doesnt seem to matter, it either expresses completely or there is nothing. the cells also do grow, i always check the OD at the end of induction and in the small culture, the cells were 0.6 OD when induced, and 1.2 OD when harvested. i have had situations where they are only 0.62 OD when i harvest (from a 0.6OD induction) and these never work, but it is inconsistent. sometimes the cells grow and sometimes they don't and i can't see a pattern. 

i am not using pLysS strains because i was getting worse expression there. I am using NEB BL21 DE3. 

the protein is around 20kDa, so it is small but when i have had good expression, it is a whopping band on the gel and very very obvious. i always run a 0hr and 4hr induction from a good prep as a control on my gels to compare. the protein does go to inclusion bodies, it is quite insoluble but the purification is a denaturing one that takes advantage of this so it is OK for me. i don't know why the bacteria would just stop expressing it. 

i have asked everyone i know and tried retransforming in commercial cells but i have not had much luck. if the protein were toxic, i does not make sense that sometimes it would express so great and other times not at all. it would always be toxic... 

in the past i have tried to optimize IPTG and temperature, 18 degrees overnight, 20, 24, 30 for a few hours or overnight (and every combination) and the best expression was always at 37 for 4 hours. also it was never a problem. really last summer i made this protein a dozen times, only now is it behaving this way. i had the original plasmid and glycerol stock but they did not express consistently, which is why i re-cloned it, but to only partial success again.

i appreciate any suggestions!! thank you for reading!! 

You say that you kept a glycerol stock of cells for expression? Only keep a glycerol stock of your plasmid in a cloning strain of E.coli never in an expression strain! If you only have a glycerol in an expression strain and no original mini you can just about prep from the expression e.coli, re-sequence it  and re-transform into a cloning strain for glycerol archiving.

For expression you will get much more consistent results transforming each time-your plates will be good for expression for a week or so in 'B' strains on plates with glucose.

Now that you say that you have strange growth patterns/low final ODs I am a bit concerned that you may have a phage problem, especially if anyone else in the lab is also  having similar problem:if  that is the case you will rarely get expression from your cultures and it can give you this complete hit or miss effect with your cultures

If you streak or re-transform are your colonies round or do the edges look a bit uneven? Try a phage resistant cloning  strain and a phage resistant expression strain.

thank you for replying!

no one else in our lab does this sort of expression. after i did not get expression from the glycerol stock, i did exactly what you said and retransformed from my original clone - i had the plasmid frozen also. but this still did not work. so i re-cloned it, sequenced, transformed into BL21 fresh, and did an induction test in 5ml - this worked great! but then the next day i took the same colony and went up to 500ml and there was no induction, same conditions otherwise.

what do you think? 

We have been doing expressions and scale up for quite some time at our university.

For some reason, we did not like the BL21 host for expression as we faced a lot of issues in the same.

I would advise you to try expressing the same in a GJ1158 system, it uses NACL induction and gives good quality expression.

Scale up is also not affected in this system and we have scaled up proteins like rWBALT, rWBSXP etc to fermentor systems also.

I dont think trying this would hurt your time/process. Just get the strain and transform and do the expression side by side with your BL21 expression studies.

Regards & Good Luck..!!

Hi there,

According to what you wrote, you managed to get something in a first expression experiment and then the day after starting from the same clone it didn't work anymore. So it might that the transformed cell is very unstable from a plasmid point of view. I would suggest to start your 500mL culture with an overnight culture obtained directly from transformation performed the day before. In this case the preculture contains fresh cells resulting from different transformation events (expression level in BL21 does vary a lot from one clone to an other so working this way does make the average between clones).

Have you considered aeration efficiency? I newer go more than 0.5L medium in 2 L Erlenmayer flask. If I know protein to be tricky I even go 0.25 L medium/flask

hi 

i agree with the logic of Dominique Liger .....

Hello and thank you for your comments!

I did try a fresh transformation straight into the 500ml culture, unfortunately it didnt work :( 

I also have a lot of aeration, 500ml in a 2L flask, so that also cannot be the problem.

The most bizarre thing is that it does work sometimes, I have gotten good expression at least 3 times in all this, but there appears to be no consistency as to why. As soon as I get good expression, I scale up and then it doesn't work. Then I try again in the small scale and that doesn't work either. It is very frustrating. Then I tried to retransform and reclone, and I will get one good expression, but it doesn't repeat even with the exact same conditions and the exact same clone.

Thanks for the suggestion of a different bacterial system, I will look into this. The strange thing is that I have expressed liters and liters of this in the past (the last time being last summer 7/15) and then I had protein for a while. But now when I am trying to make it again, it just won't express, despite being the exact same plasmid and system. 

Thanks again for the comments. Any other suggestions are greatly appreciated!

Hi,

Did you try the addition of glucose? I have seen proteins with no expression rise to 10s of mgs/litre just because of the addition of glucose.

Have you tried a 'phage resistant host? 'Phage will give you really inconsistent growth  and/or expression that you may not spot unless you know what you are looking for. In my old lab we spent months of inconsistent/repetitive failures in growth and expression befpre switching to 'phage resistant strains. Especially important for a lab that accepts samples from many different sources.

Thanks for the comments!

Phage resistant sounds like a good idea, but I think the cells I am using are already resistant to at least one kind of phage:

https://www.neb.com/products/c2527-bl21de3-competent-e-coli

Is there a cell strain you would recommend for this? Are there other sorts of phage out there? We do not receive many samples, in fact no one else in the lab does bacterial protein expression, but others have been having some issues just with growing bacteria for a maxiprep, so a phage floating around would not be surprising. Would spraying down the incubator with 10% bleach help that?

Glucose is also a reasonable idea except that why would it grow sometimes without anything extra? If the cells needed glucose I would think they would always need it... I have noticed that the times I get good expression, the cells grow at least 3 fold over the course of induction, and the times that I don't get expression, they barely grow. So certainly the growth is related to good expression, but I don't know why it is so variable.

You are right these NEB cells are T1 phage resistant and the the particular mutation they have (fhuA2) also confers resistance to other phage strains (e.g. it affects the same protein as the tonA [I think] mutation in Omnimax2 cells that confers T1, 3, 5... resistance). It is possible that you have a different phage strain present in your lab, especially when you cannot grow cells for plasmid preps. If it is phage you can try cleaning but it will undoubtedly recur unless you take away the phage host-i.e. use only resistant strains for everything in your lab. I made some Rosetta (DE3) LysS and B834(DE3) 'phage resistant back in Oxford (OPPF) that they may be able to share with you as did SGC in Oxford.

The explanation of the effect of glucose was in my first answer-it can block expression of toxic proteins until you have  a good biomass/induce with IPTG. So leaky expression can block growth and reduce plasmid stability but I would expect the results to be more consistently bad in the absence of glucose. Try it it is so simple!

Hi Daphne,

I am having the exact same problem. Could you solve the problem. Also, in one comment Nick asked about the colony shape. How to know if I have phage problem or not?

hi

this problem have taken place for me a few times. I recommend you change host  and use of new host of the same strain. certainly it will be solved. 

Is your 500-mL culture in 1- or 2-L Erlenmeyer flask? Use a 2-L flask so you have a lot of aeration. Have at least 75-80% empty volume in the flask.

I had the exact same problem. I read Nick’s suggestion here and he is right. It was a phage contamination. my cells were phage contaminated and we had to throw away all the Rossetta cells we had. New cells works perfect.

Mahsa Shirani how did you figure out it was phage contamination?

The easiest and fastest approach is use of a fresh E. coli host strain.