Try retransforming your plasmid and picking a fresh colony. If you have an Amp-resistant plasmid, try using a more stable version of the antibiotic, such as carbenicillin.

What is your growth OD? between 0.6-1?

Upload protocol you using, every step is important, then it would be easy to decide

Try retransforming your plasmid and picking a fresh colony. If you have an Amp-resistant plasmid, try using a more stable version of the antibiotic, such as carbenicillin.

All of the above answers are good

Always inoculate with a colony from a freshly streaked plate

Use 2X strength LB media and check the pH before you autoclave

If you are using Amp as you selection marker use more

If you are doing overnight starter cultures use 400ug/mL Amp and used 300 to 400ug/ mL Amp during expression

Use 300 rpm on the incubator and never fill your flasks to any more than 1/3

If you are doing overnight expression use low temp ie 20C

You should avoid induction at OD600 > 1 if you can.

Hi, I would add to above answers also: try induction around OD600=0,6. Do not let the culture overgrowth! It is good hint with lower temperature ON ( I use even 18C). If you are expressing overnight you could also try auto-inducing medium instead LB. You could also try with BL21(DE3)RIL, if available. They usually give higher yields, but remember about chloramphenicol resistance.

Agree, all answers are important to you. I would still like to ad:

make a trial (of course using a freshly transformed colony!) different growth temperatures, different IPTG conentrations, different induction times. It is once doing a broader overview. You can immediately spot the optimal conditions. I even would include two, three different E. coli strains (as mentioned above the RIL, but also Rosetta or so).

And certainly, as you admit yourself - an E. coli colony on a plate is not a very good storage option for a strain with any plasmid, especially not an expression plasmid.

Good luck!

As Vladimir noted, I think we need a little more information to assess the problem, but there are many reasons possible, some of which you noted. I would like to know what is the plasmid/promoter system being used. Is this really a lacUV5 or Tac or other lac-derived promoter on the plasmid or is it really BL21(DE3) used with a T7 promoter plasmid? For the latter, 1 mM IPTG would likely be too high. I agree with Daniel that it is often necessary to retransform every time, and Ted's tips are also good, although the temperature depends on the protein to some extent.

Dear Arianna,

I would start with new transformation of BL21 (if you have initial plasmids). Then use up to 5 colonies for inoculation of individual minicultures and grow overnight at 37 C). Measure od of each and make glycerol stocks from two with highest OD. Then use these for your large scale expression.

I do not think that your sonication method is incorrect (it's never the case) and IPTG concentration seems to be also ok (hey it worked at this concentration before right?).

To keep your E. coli happy you can switch from standard LB to very rich NZY broth.

E. coli also like nicely oxigenated media so increase shaking speed to 180 rpm and higher if you can.

Last option is simply expressing your cells in larger volumes.

Good luck

It may be a bit trivial, but make sure you're growing the 500ml in an 1.5L flask (at least!). The bacteria need plenty of oxygen to grow properly and express the protein.

Also, you can use the BL21RIPL strain. sometimes it yields more protein.

The suggestions above are all excellent. One other thing is that if you are using 500 mL expression culture, use a flask with at least 2L capacity to get maximum aeration. If possible, use a 'baffled' flask to get even more aeration.

In agreement with other correspondents, I stress that you must use a freshly transformed colony. Not a good idea to store glycerol stocks of expression plasmids in BL21 - it's sometimes ok but mostly not and is banned in my lab as bad practice!

If you cannot be sure that the BL21 strain you are using is pure culture buy in fresh competent cells or glycerol stocks. If your BL21 stock is contaminated with another coli strain without T7 apparatus then you'll see no, or hugely reduced, expression.

Hi..your IPTG is fine but what is your induction time? In general for His-tagged proteins, overnight induction at 16C works best to get good amount of protein. Fresh transformation and right amount + quality of antibiotic is a must.

If you already purified your protein before using the same protocol, we can exclude proteolysis/aggregation (...) of your protein during growth of your culture.

One of the possibility is the sonication, as you mentioned. Hence, you might want to follow every steps of your purification by Western-Blot. You can also combine lysis with lysosome (freezing/thawing cycles) and sonication.

Another possibility is the (partial) loss of your plasmid. This sometimes occur when inoculating your culture with your preculture. To avoid this phenomenom, perform directly your culture (no preculture) with either a colony or a fresh transformation (I am always using this last option).

Good luck.

Joachim Gerhold ,why an E. coli colony on a plate is not a very good storage option for a strain with any plasmid, especially for an expression plasmid.

As the plasmid gets lost with time, so it is best to do fresh transformation

Hi, Although you got many comments but still I like to add something..I have experienced all these...

No. 1) We must use freshly transformed E.coli

No. 2) The IPTG concentration is very high (not too low as u mentioned!!)

No. 3) Please check the solubility of your proteins...if it is not soluble then you need to optimize the condition or need to change the stran!!!

Good luck!!

There are so many good tipps up there, but since you seem to have a system that´s working well, I´d propose the following strategy:

1. retransform BL21 with fresh plasmid. I´d not go as far as transforming everytime you want to overexpress; if you stock away several BL21 transformants directly in glycerol and then test which one is experessing your protein best, you are kind of safe if you always start from that glycerol stock (NEVER use a colony from an old plate that´s been sitting in your fridge for days; why? BL21 lacks the recA1 mutation (gene for recombination) and can do funny things to your precious plasmid. Therefore, all cloning etc in DH5alpha or similar; BL21 only for overexpression).

2. next I´d check expression and yield in 250 mL cultures with exactly the protocol you used before, because you had high yield with that. If this high yield is reproducible and you just want to go up to 500 mL culture volume, I´d make it simple for myself and just grow two flasks with 250mL where I know my protocol works fine. If you want to go up to 500 mL in one flask, you´ll have to fiddle with IPTG, starting inoculum etc (see tips above) which will waste your time.

3. if you can´t reproduce good yield with 250 mL, figure out if you did eveerything exactly as before. If so you´ll have to start trouble shooting going through all the tipps above....antibiotics conc., IPTG conc., temperature, OD600 at induction, resequence plasmid, check bacterial strain - if it´s an "old" lab-strain try to get another "fresher" BL21 strain from another group, ... .

Good luck!

Huma Saleem: A colony on a plate is biologically active, meaning, cells replicate and the plasmids will be replicated as well. Each replication bears the risk of introducing mutations into your bacteria´s genome and plasmid. Both can impact protein expression efficiency. Additionally, if you get mutations in your expression plasmid they can either affect your promoter (making it less efficient-> less protein expression as the result) or even worse, if they occur in your genes coding sequence they might not be silent mutations and result in changes of the amino acid sequence...you´ll overexpress a mutated protein without knowing it.

In expression strains such as BL21 recombinase genes such as recA1 are not mutated, as they are in library strains such as DH5alpha. This additionally increases the risk that your plasmid construct undergoes "funny" recombination resulting in either duplications or deletions, depending on what happens exactly.

All in all, you want to minimize any of the above to happen as you want to keep your plasmid and bacterial strain (genome) as conserved as possible so you can reliably reproduce experiments. In glycerol stocks at -80°C the above processes don´t occur, so it´s the best way to store your bacteria. Make glycerol stocks as soon after transformation as possible. Also, keep isolated plasmid stored away as well as an additional backup.

Hi Ariana, you can also try to use BL21 Stars, it is very good for overexpression . Good luck, Tetyana

Lots of good suggestions about re-transforming fresh cells and using carbenicillin. One problem with scaling up is that the cells are producing beta lactamase which is secreted into the media and breaks down the antibiotic. The longer the culture is grown in the same media the more beta lactamase present and the faster the antibiotic will be degraded. Once the antibiotic is gone the cells do not need the plasmid and will get rid of it, especially if it expresses a toxic protein.

If your expression worked in a 250 mL culture, then a 500 mL culture should work fine too.

Here is my protocol:

Transform cells and plate on antibiotic containing plates (make sure antibiotic stock is "reasonably" fresh and vortexed before using if stored at -20oC, amp/carb will settle as a clear phase at the very bottom of a thawed tube leaving water on top). The next day, once I see colonies, I will take the plate out of the 37oC incubator and leave at R/T (this slows down the growth of non-recombinant satellite colonies) and wait until 6-8 pm to pick a colony off of the plate and start a 5 mL O/N culture at 37oC with fresh antibiotic at 300 rpm. The next morning I will spin down 1 mL of the 5 mL O/N culture and remove all media, then resuspend that pellet in 1 mL of fresh media and use ~100 uL of that to start a 50 mL culture in prewarmed media with fresh antibiotic at 300 rpm. That culture should grow up to OD ~0.3 (595 nm) within 2-3 hrs. When it reaches ~ OD 0.3-4, I spin it down, carefully remove the media (which contains beta lactamase), and resuspend in 5 mL of fresh media and add to 0.5-1 L of prewarmed media at 37oC with antibiotic, and antifoam A (Sigma), in a baffled shaker flask (sized for the volume of media, a 2 L flask for 500 mL of culture) at ~300 rpm (make sure cap is properly vented, they will need lots of oxygen). That culture should grow up to OD 0.3 within ~3-5 hrs. Right before I add IPTG, I add another dose of freshly made antibiotic at the original concentration (so if you used 100 ug/mL of Amp to begin with, add another 100 ug/mL). This keeps the beta lactamase already present in the media busy, since is has degraded most of the original antibiotic, this forces the cells to keep the plasmid. After 3-4 hr at 37oC I then harvest the cells.

Sometimes I've had very different results with different colonies, so that can be an important issue. As many have suggested you should standardize the conditions comparing yield and solubility taking at least 2 or 3 freshly transformed and try a couple of IPTG concentrations (you could try lower than 1mM) and 2 temperatures (30 and 37°C) and times for induction. If you protein is stable you can try overnight incubation at low temperature and IPTG concentration. If not, I do not recommend it. Using 5ml tubes works out fine, then lyse your cells and analyze the soluble fraction by SDS-PAGE and western blotting. According to the results you can determine which other parameters are worth trying. Then you can make glycerol stock of your colonies and use the same colony and conditions for some time reproducing the result.

You can try to retransform into fresh Bl21 and do not store them on plate but make frozen stack and plate fresh before purification. You can try different cell lines. 1mM IPTG is more then enough, it works like on/off switch so once you induce it cell will be making mRNA and proteins.

On the other hand if you have set up protocol for 250ml try to repeat it. If it works just make 2x250ml cultures instead of 500ml. I have worked with similar problem before, at the end I was expressing in smaller volumes, ON at 22C. Whatever works to get your protein!!!

If I had your problem, I would do the following way. I already know the expression and purification conditions, so;

I will do a transformation to same BL21 strain (as its already working). Next day I will screen 3-5 colonies for expression (Because not all colonies express equally). Based on SDS-PAGE results I will choose 1-2 colonies. Make a fresh o/n culture with one of the colony, and in the morning I will make glycerol stock (store for further use) with 1 ml and use remaining as starter culture. Your expression is already standardized so I wont change a lot but you can always bring down the temperature if you are using 37C as it helps. Using old plate is not a great idea.

Note: My lab mate said she has to do transformation every time to get good expression, as her glycerol stocks gave weak expression. I dont have first hand experience and dont have reason to believe why it is but worth considering.

For sonication, please check the manual to keep settings in way to keep the temperature of lysate as minimal as possible (may be below 10C). This is very critical for sensitive proteins.

if you had not purified this protein already they you should try different options what others have suggested. There are some awesome tips from others and incorporate all those to get a best protocol.

Good luck

As for your IPTG, is it a freshly made stock from powder? IPTG is heat labile, I think I always stored the powder at -20oC and always made up fresh stocks for large scale expressions.

Upscale from 250 to 500ml?

You just use 2 flasks, so that you can conduct induction in the same way you did before. You might get a wrong clone, but it does not matter whether you use glycerol stocks or new transformants. In fact, glycerol stock could go bad. Because of this, you have to plate them on LB agar and take several clone.

New transformation or colony-pick up from glycerol stock. Either way, I would suggest that you pick-up several clones and do small scale induction to check expression to use for 250ml scale. Or you just pick up 4 to 6 clones and grow each of them in 250ml media, and take good ones.

And don't try to change the condition you did before.

Well, the first thing to do is to check whether you lose protein into inclusion bodies. I remember working with E coli BL21 and there were days when i lost all my protein to inclusion bodies!! In such case, maybe you could consider induction for longer times at lower temperatures. Meanwhile, it is worthwhile to transform freshly prepared competent cells with the plasmid and pick a few colonies and make a masterplate, and analyse each of them. You could prepare a glycerol of the one that induces the best. I think sonication is not a scable unit operation and hence it maybe better to move to carrying out cell lysis using a French Press (1200 psi, 3 cycles) should be enough to lyse most of your bacteria, but in case you want to continue using sonication take care to make sure that you have your cell suspension on ice and do not generate any foam since it almost always destroys proteins. 1 mM IPTG concentration is pretty high for induction, and i suggest you keep it constant or even lower it.

Thank you all so much for your answers. They are very helpful!

I will try the process again taking into account all your suggestions,

Thanks again!

BL21 star do not keep the plasmid for a long period of time. If you are wanting to store plasmid it is best to store them in DH5alpha E.coli cells. I wold use plasmid to retransform BL21 Ecoli cells. We have had to do that recently. You are not alone.

I recommend that you use fresh transformed cells. You may not have to use glycerol stock.

Sonication generates lots of heat, thus, you should ensure that temperature is not going very high.

You are using high concentration of IPTG, you don't have to increase that.

To find out the problem, you need to run SDS-PAGE to check whether protein has expressed or not. Take out aliquot after each sample and run on SDA-PAGE. This will surely tell you, which step is affecting final yield of protein.

Soumyaditya,

For the overincubation, are you talking about overincubating the starter culture or overincubating the 250mL after I have added IPTG?

How long would you say I should grow a starter culture of 5mL?

Thank you!

Thank you!

I will pay attention to this!

Hi Arianna,

I was wondering what strain of BL21 cells you use? I have found that the high efficiency BL21-RIL (codon plus) (from stratagene) line produces the highest amount of protein yield after induction and purification (as compared to the standard BL21) because this strain also contains a chloramphenicol selection (so you could only use if your target plasmid was amp or something else). I start off with transformation and plating onto a double selection plate, select one colony and grown in 5ml 2xYT (+ antibiotics) overnight 37C (~16hr) 225rpm. From this 5ml culture, I suggest to make a glycerol stock so that for future trails, you do not have to re-do the transformation. Meaning if you have transformed glycerol stocks you can skip this step and simply add 20µl to a 5ml culture (will save you a entire day)

Pour the entire 5ml colony into 500ml 2x YT, grow @ 37C to OD600 ~ 0.7 @ 225rpm. Induce with IPTG (final conc. of 0.4mM) and add fresh antibiotics. Induce overnight at room temperature for 16 hrs 240rpm shake. As far as sonication goes I suggest sonicate on ice for a total of 3 minutes: 30sec on, 30sec rest (start at level 5 and slowly increase). Alternatively if you have access to a french press, sometimes this can work better for some proteins: 2 passes at 1.5 ml/min @ 4C.

It will be better if you use fresh transformed cells.

Optimize IPTG concentration.

Use less number of sonication cycle.

Make aliquot after each sample and run on SDA-PAGE.

This will surely tell you, which step is affecting final yield of protein.

Hi Arianna, I would like to know the concentration of protein that you are getting from E.coli cells. There are some strains of E.coli such as BL21-RIPL which are very good for protein expression and purification. Also, it is possible that using 1mM IPTG is a very high concentration and it could being toxic to the cell. I would suggest you to lower the concentration to 0.1 to 0.5 mM. Also, you can try to express your protein at a lower temperature like 25 or 30 C in case you are currently doing it at 37 C.

hi, if all the induce condition seems no positive improvement. I will suggest considering check the gene code whether very rare amino exist or not. if yes, you should change the host, Rosetta maybe a chocie. another situation, maybe use Yeast or cell expression system will be a right choice. Some gene just can't received good expression in E.coli.

If you are starting from the frozen stock, try growing the colony on agar plates, and then do the liquid culture.

sorry, first of all are you using T7 driven prot expression (pET or derivative), if yes, you should use only BL21 (DE3) Cells or derivatives...BL21 cells will NEVER allows you to express a T7 driven construct (if you do not underestand why ...read Studier, F.W. and B.A. Moffatt. 1986. Use of bacteriophage T7 RNA polymerase to direct

selective high-level expression of cloned genes. J. Mol. Biol. 189:113-130.)

if using a tac driven expression, you can use whatever strain TOP10;DH5 etc etc...the main reason for using BL21 (DE3) is because theu are protease minus. you you do nit experience proteolysis pb, go with top10!

many hints have been given here (fresh transfo, mid log, rich mediulm etc etc)

I know, one has to read the basic...but, latter it save time...good luck

Do fresh transformation of your construct before inoculation, ur plasmid could be toxic and hence might be getting lost with time.

-My IPTG concentration is too low (I currently use [1mM]).

just for the records ..T7 expression is already turn on in BL21DE3 with IPRG as low as 1micromolar:-)

what is the MW of the protein, ecoli system is not very reliable when the MW exceeds 60kD. also did you tested that the protein is not a inclusion body.

One frequent issue when scaling up is the oxygen supply getting worse at 'larger' scale. I am not sure what reactor you use for your experiments. If you are using shake flasks try to increase the shaking frequency and /or use a bigger flask. Further the coli may produce more acetate inhibiting growth. good luck!

Hi,

It's not usual practice to store glycerol stocks of plasmid using BL21 cells lines due to instability (which is possibly why you see lower expression), so I would agree with other comments to re-transform fresh each time using this strain. If we do make glycerol stocks we use XL1Blue or similar (Top10) and freeze at -80C in 10-20% glycerol.

good luck

I know it's very late now, but just in case someone else is in the same situation:

A couple of years ago, we were working with E. coli BL21 DE3 Codon Plus trying to optimize the expression of a recombinant protein. We performed a Design of Experiments manipulating the Induction OD, IPTG concentration and growth Temperature. Results shown an increase in the yield for the recombinant protein production from 50% to 77%, where the optimal growth conditions were Induction OD 0.5, growth Temperature 37°C and 1,5mM IPTG concentration, which was the most significative factor in the response surface methodology (RSM).

Good Luck! :)

BL21-Gold(DE3) competent cells are designed for easy induction and high-level protein expression.

Sharad Purohit where is the reference for that?@Sharad Purohit