If it's there before induction, and does not increase in intensity during induction, how can you be sure that this band is your protein and not some other coli protein that runs at that weight?  

Firstly, a good, clear and concise question with the pictures to help with answering it. Depending on how much protein you need you can either spend ages optimising the production (such as using terrific broth, inducing using autoinduction media, change temperatures etc) or just scale up what you have already done and hope it gives you enough.

You should not have any protein at 0hrs as  BL21 generally gives no background expression. If that is indeed your protein overlaying with a native protein, and the increase seems reasonable, then your next step is a trial purification to confirm this is correct. It is probably better to lower the growth temperature of your cells to 30C 30 mins before adding the IPTG as this helps protein solubility. Your protein expresses well after 4 hours so this is a good time to choose.

For a trial purification grow a 50 ml culture and take a 1ml sample 4 hours after induction. Lyse the cells in buffer containing protease inhibitors, DNA and lysozyme and centrifuge out insoluble material. To the supernatant add around 100uL of NiNTA beads and centrifuge (gently). Wash with a buffer containing 30mM imidazole (I use 50 mM Tris pH 8.0, 500 mM NaCl). Then elute with a small volume (50uL) of 500 mM imidazole in buffer and run this on a gel. This should have purified protein if you are expressing well. Also run the insoluble fraction, the total cells and un-induced cells to check it is expressed solubly. If this goes well then you can do a large scale expression and purification with confidence. 

Hi Matthew

It could be due to the fact that T7 is know for a leaky promoter. I am doing a western blot right now and going to use Histag to determine if the band is my protein or not. Any suggestion?

Thank you Ellis, I will try to change the temperature. Do you have any idea why there was a band there at 0 hour beside leaky promoter?  

Possibly there is just a protein that size produced in E.coli. Maybe you can check a strain with a different plasmid/insert. Is there a reactive band in your blot?

I am running a blot today. I should be able to let you know by this weekend. 

Most cases of detectable basal expression in T7 systems are caused by runoff transcription from upstream antibiotic resistance genes or traces of lactose on the culture medium. In the first case the only solution is to sublone to a vector where the selectable marker is transcribed away from the target gene; in the second, try adding glucose at 0.2-2% or checking different batches/suppliers of tryptone, casein hydrolisate, etc.

In expression experiments I always include a control culture of the same host strain, but transformed with the empty vector, to avoid mistaking a host contaminant for 'my' protein.

One other thing I forgot to mention in my original question is that during the OD600 measurement. What do you guys think about the OD's values? Are they reasonable? Can I do anything to improve it? Thanks

Hour          OD600   (1 mM IPTG)

0hr              0.44

1hr              0.53

2hrs            0.55

3hrs            0.55

4hrs            0.60

12hrs          0.90

13hrs          0.91

14hrs          1

15hrs          1.2

I see you are using pLys. Shouldn't that help reduce production of protein before induction? Are you adding the antibiotics to maintain the pLys plasmid? The OD looks ok. I wouldn't express any protein at 37C for more than 5 hours. 

Hi Tamjeed, 

Do you think I should low my temperature from 37C to 30C?

Hard to say with the data Lewis. As Ellis commented above, you are better off lowering the temperature, inducing and purifying the protein. You can get an idea of how much soluble protein you get. If thats enough for your experiments you are all set. If not, there are a whole host of things to tinker with.  Good luck!

Run as a control BL21 (DE3)plysS without your plasmid, if this band is still there - most likely no expression. If so, try different strains - check their phenotype for guidance. If protein is eukaryotic - try codon optimization or use  Rosetta™ , Rosetta™ 2... competent cells for expression.

As already mentioned there are many things that can be tried and optimization take time. Looking carefully at the gel picture I do think there is some increase between 0 and 2h (thought little) and the idea that a native E. coli is in the same size make sense. Here are a few simple options:

Repeat the growth curve with vs. w/o IPTG. If there is a difference than probably your protein is expressed. If not, hard to say, either no expression or it does not effect growth.

As suggested, run the E. coli without your plasmid, or even better with empty vector plasmid.

Make sure your protein is not in the inclusion bodies. After sonication take the pellet, resuspend in loading buffer and heat for ~10 minutes. Centrifuge again and run the soup.

Try lowering the temperature. I have used as low as 18C.

Try lowering IPTG concentration, 1mM is no little amount.

Yoram

how did you know it is your protein, first you have to check through one easy method to cut the band from gel and send for edman sequencing you will get the first 15 N terminal amino acids. may be it is another protein which is similar to yours MWt you have to confirm first. 

if you confirm and the band still appear at the zero induction, it depends on your plasmid which plasmid did you use? because I think it could be a background expression. 

The ODs do not look good, by 2 hr it should at least double and for nontoxic proteins I get 2.0 ODs by 3-5 hrs (if you are expressing a toxic protein I would use BL21 pLysY Ip, New England Biolabs). Are the cultures aerated?, when I add IPTG I partially open the flask covers (I use aluminum foil) so oxygen can freely get in. Protein synthesis needs oxygen. Are you using a rich media?, need that for good protein expression too (2XYT and plenty others).

You can try expressing this protein at low temperature and also you can try inducing your bacterial culture at different OD. Sometimes that also can affect the expression drastically. 

While looking at the gel , it seems that there is no over-expression of the cloned gene.The most probable explanation is that the conditions for over expression have not been optimized/determined. It is a result of a single shot of IPTG at 1mM Conc while the bacteria needs to be titrated against a range of IPTG concentrations to determine optimal overexpression level. Lowering of temperature can also be helpful.

best of luck

I would definitively add a negative control of your strain without the plasmid in order to check if this band is still there and it would also be so so helpful tu perform a western-blot with an anti-His antibody in order to detect if this band actually correspond to your protein and if it is present in the no inducted cells.

Hi Everyone, 

I would like to give some update on my protein expression problems. 

1. I ran the my previous sample from the picture above on a western blot. The idea behind this to see if I can detect my protein by using Histag antibody. The result from the WB did not show any band at all. Not it raise another question about the antibody quality. Due to the WB result, I decided to regrow a new colony from my freezer stock and changed the expression temperature (attached picture). 

Now I have several questions regarding to the result from this experiment.

1. Why am I seeing the expected band at Zero hours without IPTG?

2. What do you guys think about the band after overnight at 25C with IPTG?

3. What should I do from here? 

Thank you so much for your time and have a great day.

You can try doing Ni-NTA column chromatography with cell lysate to see if your protein is actually getting expressed. The idea is if your protein has His-tag and is getting expressed, it should get eluted at higher imidazole concentration. You can give few washes with low imidazole concentration like 10mM and 20mM and then elute with 250 or 300mM imidazole. Since the expression seems to be pretty low, you can concentrate the fraction eluted with high imidazole concentration.

P.S. I have used BL21(DE3)pLysS strain and I do see a band around 25 KDa. So, that could also be E. Coli protein. You will know after Ni-NTA. 

Hi Lewis,

You can check protein expression by running a western blot using anti-His antibody. Sometimes, it is difficult to detect expressed protein using SDS-PAGE gel especially if the protein is not highly expressed.