Especially with clones expressing toxic proteins it is very very common that you get an isolate with a mutation. If expression is toxic there will be a very strong selection for a clone that no longer expresses. I would consider retransformation of your DNA into your BL21 strain and checking a number of independent clones, and note especially if you have larger and smaller colonies.

Did you check on a western that your protein is not expressed? Toxic proteins are often put into inclusion bodies and are not soluble. You might find it in the cell pellet.

If there is lactose in your culture, it should grow slower from the beginning. It might help to grow the cells at lower temperatures (I have done this at 18°C for a membrane protein), which takes then much longer to reach the final density.

Thank you, Christian for suggestions. I have checked the presence of the protein in the whole cell lysates, so if my protein would go to the inclusion bodies I would see it. Similar protein is not detectable by western-blot. I think that glucose might inhibit the lactose action. May be glucose is sufficient to inhibit IPTG too...

I had the same problem for certain GST tagged proteins but not for all GST tagged proteins. I used DH5a strain. I would not suggest glucose or lactose could be the problem and would not consider the bacterial strain as a problem. If it is so, it must be same for all constructs. For me it is highly construct specific problem and the growth is super fast after addition of 1mM IPTG. I am sure someone have an answer for this..!! Waiting..!!

Hmm... Currently, I'm also checking the idea that mRNA encoding for heterologous protein may have a secondary or tertiary structure that might inhibit protein translation.

If your protein is not visible in whole cell lysates (which then also include inclusion bodies) than I think its not expressed. How big is the protein you want to express?

I would try to optimize the conditions, meaning varying the IPTG concentration as well as the growing temperatures (32, 27, 20...).

My protein is small, about 30 kDa. Earlier in other lab it was successfully expressed in E.coli cells in similar expression system with T7 polymerase.

Especially with clones expressing toxic proteins it is very very common that you get an isolate with a mutation. If expression is toxic there will be a very strong selection for a clone that no longer expresses. I would consider retransformation of your DNA into your BL21 strain and checking a number of independent clones, and note especially if you have larger and smaller colonies.

Indeed, I have red that on LB due to some background level of protein expression might allow to grow colonies with reduced expression of protein. If this true, the bad expressing plasmids might be selected already on the cloning steps (!).

In my experience LB cannot induce suficient expressiom and in case of toxic protein leaky expression you will get very low densities to start with. Please ensure you are not picking a wt or mutant colony. One way to quick check this is by modified colony PCR . Take 1ML culture prior to induction extract plasmid DNA and use this as template with your protein specific primers.

In protein expression experiments I transform BL21 with plasmids that were checked by sequencing. But I have checked only protein encoding part and about 20-40 bp of vector sequence that flank the insert. No mutations were found. For sequencing I used T7 primer that anneal on T7 promoter. I guess if T7 promoter was somehow mutated, then I would get very poor (i.e. weak signal and short reading) or no sequencing results. They were very good.

Hello, it is unusual that the LB inhibits the induction with IPTG. I usually work with proteins of different sizes. Some are toxic and expressed little. The solution, for me, has always been used C41 (DE3) and C43 (DE3). I enclose a job in which these bacteria tested. http://www.ncbi.nlm.nih.gov/pubmed/15294299

This is recurring problem. If you have never seen expression you may simple have a problem with your construct, cells and/or reagents. If such a problem is discarded you need to check expression on newly transformed BL21 cells. You might consider doing the following : 1. Transform BL21(DE3)pLysS cells (make sure you have the right cells) the evening before you check for expresion 2. In the morning, pick single colonies and put them in 3 ml LB. 3. Let them grow until OD 0.7-0.9 and induce protein expression with 1mM IPTG. 4. Grow them for 2-3 extra hours and check protein expression in a total lysate. As a control, you should do the same with a construct that you were able to see expression in the past.

It sounds like your problem arises from the toxicity of your protein, which is likely to select low- or non-producnig segregants. These may not be necessarily due to mutations of the gene (you would still be able to detect the polypeptide) or its promoter; for example mutations affecting the expression or activity of the polymerase would yield the same result. I would recommend that you use a host with a more stingent control of recombinant gene expression, such as the arabinose-inducible strain BL21AI available from Invitrogen. Don't forget that if the natural isomer of hexoses is usually the D form, the arabinose used for induction is L-arabinose. Also, since your expresion plasmid contains a lac operator, add IPTG together with arabinose for induction

Another option would be to use auto-induced expression and test different aeration to control induction of expression. Everything is detailed in this paper: Studier, Protein Expression and Purification, 41 (2005).

HPH and good luck

One more trick. Begin with 100 ul/ml ampicillin and add fresh amp after IPTG induction (+ 50 ul/ml). Alternatively use carbenicillin instead of amp

Pedros approach appears the right one..!! I will test it. I don't think toxicity is a issue because the bacteria grow in mass several fold than GST-fusion expressing batches. All we need is the fusion protein expression and if LB broth induces leaky expression, it should be fine. But the problem is no protein expression but great bacterial growth. That is the issue. So I wouldn't blame LB broth. Running out of Ampicilin is one potential cause I think (after reading Pedro's comment).

The overgrowth may exhaust IPTG and Ampicillin, a likely cause..!!

Hello,

I never heard nor read that LB could inhibit the expression. in case of toxicity of the target protein, the T7 expression system is not the best because it is leaky (it is inherent to this system) and the addition of pLysS is not sufficient to close it tightly; you may repress the T7 RNA polymerase expression before induction by cotransforming with pLysY (stronger than pLysS and not detrimental for the bacterial cell wall) but in case of failure, I think that the pBAD expression system is better because it is almost completely closed in absence of inducer (L-arabinose) and the expression is inducer dose-dependent, so you can modulate it and avoid a counterselection of producers.

good luck,

Colette

You have probably lost the plasmid. Amp is s poor selective marker especially for toxic genes because plasmids make so much lactamase that all of the amp is destroyed early in culture growth and then the plasmid can be lost without penalty. See Studier's paper in Methods Enzymol.PMID: 2199796. Best to switch to a kanR pET vector.

At last, I have managed to express my protein. The clear band with predicted mass has appeared after induction on modified medium after addition 1 mM IPTG and incubation ~2.5 hours. This medium is close to TB, but instead of yeast extract I have used YNB (Yeast Nitrogen Base 6,7 g/liter). However, I suspect the major problem was the plasmid loss or mutation, since some colonies from the same plate was unable to express protein. The plasmid loss or mutation, is likely caused by leaky expression from pET vector with T7 promoter. I managed to see the slight protein band in celsl grown on LB without additional glucose under non-inducible conditions. I guess, this quantity is enough to force cells to use various ways to get rid off or mutate the plasmid. I also noticed that cell cultures that do express my protein grow very slowly under non-induced conditions, while cell cultures that do not express protein grow normally.

Now, I have encountered another protein expression problem, but this is a theme for another question.

I would like to thank all of you, for very helpful answers!!!

Dmitry,

Plate out your cultures without amp and pick colonies to plates + or - amp. You probably will see a lot of amp sensitive colonies.

A useful trick is to use ticarcillin-clavulanic acid (25 ug/ml) in place of amp. Clavulanic acid is an irreversible lactamase inhibitor so it kills off the lactamase in the medium, but not in the periplasm (can't get in).

The ticarcillin-clavulanic mixture is available from several companies. As a drug it is called Timentin. The amoxicillin-clavulanic acid mixture does not work on E. coli in my hands.

John

Thank you, John! Very nice trick!

@Dmitry: One question about your clones: How do the plates look like where you picked them? Nice and round single colonies? Or are the colonies, which are surrounded by many small dots? If the later is true, then you see satellite colonies, which can exist because of secreted beta-lacatamase and they can fool you. What helps here is to cool the media further before adding the antibiotics and add higher concentrations.

Christian, the plates contain only single colonies, no satellites, despite on the fact, that I usually add antibiotics in the hot media.

A few further tricks for toxic proteins include:

Inducing at higher OD - around 0.8-1.

Using Auto Induction Media which has a more gradual switching on and tends to yield higher cell density.

Also try using carbenicillin as it is more stable.

Dmitry,

I have the same problems as you. I could not see the clear induced band after IPTG induction. I am using pET28a, which has Kanamycin resistance gene, so it is not likely due to the antibiotic degradation. Could you please provide the revised media recipe so I can give it a try/

Thank you very much,

Jianling

Hello Dimitry, I'm having a very similar problem, the only difference is that my proteins aren't toxic. Could it be possible for you to share the recipe of your medium?

Thanks :)

Hi Joyce!

And sorry Jinaling for delay. So my TB media is the following:

- Tryptone: 12 g/l

- Yeast Nitrogen Base: 6,7 g/l

- Glycerol: 40 ml/l

- mQ to 1 liter

- adjust pH to 7.5-8.0 with KOH

I think yeast extract contains glucose which competes with IPTG for expression induction. I changed it to YNB which has all vitamins, minerals that are vital for E.coli and no other sugars.

I have found also that T7 promoter is damaged in a number of constructs that fail to express my protein. Check that your T7 promoter is not damaged by PCR with T7 primer and some primer for your gene to express. Another reason for failed induction is a damaged site for NdeI (perhaps NcoI can also be damaged). If your gene is cloned with this site (or NcoI) check that it can be excised with this enzyme. Or sequence your construct starting from upstream of the T7 promoter.

Thank you very much :) I wiil try the media, hope it works :)

Is glycerol 100%?

Yes glycerol is 100%.