GPCRs a very complex Eukaryotic membrane proteins. These are having seven trans-membrane helices and are glycosylated. So, expressing them in E. coli will be tricky. I would suggest to used Pichia system instead of E. coli system.

Santosh.

I think we need some more information here. In general, if you have problems expressing, you might want to recheck if everything is in frame and modify the temperature. Maybe try 15°C overnight to see if anything comes up there. Also, you have a TM protein, those usually are more difficult to overexpress. I found that some proteins are easier to express if you modify the N-terminal codon usage for E.coli. Again, if you explain a bit more detailed what you tried, it might be easier for us to suggest modifications. Did you use 2xYT or standard LB media? At what OD do you induce? What temperature do you grow them at, what temperature do you induce them at? How did you check the expression, how often?

GPCRs a very complex Eukaryotic membrane proteins. These are having seven trans-membrane helices and are glycosylated. So, expressing them in E. coli will be tricky. I would suggest to used Pichia system instead of E. coli system.

Santosh.

I used this protocole during my PhD work and it was working good:

The E. coli strain BL21 (DE3) codon plus was transformed with the expression plasmid. The next day, 25 ml LB-selective medium was inoculated with a single bacterial colony and grown overnight at 37°C and 190 rpm. Subsequently, 1 l of LB-selective medium inoculated with 10 ml of the ON culture and incubated at 37°C until OD600 = 0.5-0.6 was reached. Protein expression was induced by adding 1 mM of IPTG. After incubation at 14oC for 16 h the bacterial culture was transferred into a centrifuge tube and centrifuged at 4,000 x g for 15 min at 4°C. The bacterial pellet was used for protein extraction or stored at -20°C.

Good luck

In my opinion, for expression, you can try temperatures and time intervals e.g. (18-37°C) and 4 hour-overnight.

Dear Robert,

Firstly, how do you know that your protein is not being expressed? Did you confirm through western blotting?

I think, Mathias has given you some good advise on expressing your recombinant protein (ie. expressing them at lower temperature). Additionally, you can also try fusing your protein with high expression and solubility vectors like the Nus A tag, MBP, Intein mediated Chromatin, SUMO or the thioredoxin tags. These vectors will help to express and solublize your proteins.

However, like said, more information pertaining your query will help to to rule out the best option.

Cheers

One problem with expression of proteins in BL21 is that expression is quite transient and sometimes varies wildly in level. You want to aim for a happy medium; too little expression and you need litres and litres of bacteria, too much expression and you run the risk of inclusion bodies, and you cannot purify any function or correctly folded protein. I have taken the following approach whereby I transform BL21-DE3 pLysS and pick 12 colonies, then just before I leave in the evening subculture them into 1.5ml of broth and leave them overnight at room temperature shaking. The next day split them into 3 500ul cultures, put one at 4C, the other two at 37 and leave them for 5 hours. Add the IPTG to one of the replicates a incubate for 1h. Spin down the bugs and wash in PBS, re pellet and resuspend in 50ul of 1xSDS loading buffer, sonicate a lot ( and I mean A LOT), boil the samples, do not cool them, just run 10ul on an SDS page. Stain the gel with coomassie blue and you should see which clones induce the best level of expressed protein. Take the spare culture from 4C and inoculate 50ml of broth with that and leave it overnight shaking at 37C The next day add 500ml of broth and incubate until the OD is about 0.6, add the IPTG (add it as a powder, this seems to be better than a pre made solution) and incubate for 1h. Dilute the samples with ice cold PBS and centrifuge the pellet 4,000k for 30minutes at 4C, remove the supernatant and store, or use your pellet.

Generally speaking if you have really low expression you are pretty safe to grow your cultures at 37C, lower temperatures are only needed if you have inclusions and solubility issues. If your expressed protein (by SDS PAGE and Coomassie stain) is more than 10% of total protein, there may be an issue with your proteins essentially precipitating straight off the ribosome and being difficult to purify. This will be of special concern with a GPCR, bearing in mind that it is a eukaryotic membrane protein and you are trying to express it in a prokaryotic system. Your protein is also likely to be big and there are two other issues regarding eukaryotic gene expression, and that is amino acid usage and disulphide bond formation. To these ends you can use Rosetta-DE3 which contain 6 or 7 rare e-coli amino acid codons, or Origami DE3, which have mutations in thioredoxin reductase and glutathione reductase genes to aid disulphide bond formation. There is also another e-coli line call Rossetta-Gami, which is has the extra amino acid codons and enhanced disulphide bond formation.

Now if, as I suspect, you have a problem with just basal expression, this could be due to nutrition and protein synthesis, and so use 2xYT ( as Mathias suggested), SOCS or Terrific Broth to get the bugs making stuff faster, and here the Rosetta bugs may help as well. You could also try increasing the amount of IPTG you use, and add it as a powder. I suspect though that your protein will not fold well being what it is, a GPCR, and you will need to try something else such as baculovirus or Pichia as Santosh suggests.

Good luck

I have tried expressing at 37, 30, 18 for 3hrs, 4 hrs and overnight with 0.1, 0.5 and 1mM of IPTG at 0.6-0.8 OD and I use LB broth for cultivation but all have this has been futile and for confirmation of induction we run a gel and stain with CBB. At the moment we can not do a Western blot analysis for the protein as we want to mass express before injecting in animals for Ab production. The protein is 25kDa. Thank you all

Dear Robert,

Sometimes the peptide is made at OK levels but forms inclusion bodies that are occasionally so big that the peptides don't even make into the gel, so you think the bugs are not making anything. If that is the case you could try lysing the bugs in an SDS PAGE loading buffer containing 2M urea, 8% sucrose, 2%SDS with lots of sonication. Run the lysate through a qiagen qiashredder, add Bromophenol blue and mercaptoethanol to your normal concentration, boil the samples and load straight onto a gel containing 2M urea and see if they make anything. If you see the peptide you may have a bit of a problem as the protein may form a huge inclusion body, and you might have to make your mass expression lysate in upto 6M urea to solubilise it. You will then have to dialyse out the urea and hope the peptide does not precipitate. If you are trying to make an antibody, you could just make a series of nested deletions until you get fragment that expresses efficiently, and go with that.

Good Luck whatever you choose to do!!