when using E. coli BL21 pLys, grown in Terrific Broth, for toxic/non-toxic protein expression I normally induce protein expression at ~0.5 OD using IPTG, and the resulting induced cultures double ~2-3 times and then stop growing during the ~3 hr induction period. But Terrific Broth with 10-20 mM MgCl2/MgSO4 and good aeration has been reported to support cell growth up to 18 OD. Has anyone tried inducing protein expression with IPTG at higher ODs, like 5 such that the final culture is around 15 OD when harvested? Maybe the higher OD is only good for plasmid production?
UPDATE, for pET T7 expression plasmids and BL21 pLys cells, inducing at OD 1-2 for 3 hr results in a final OD of 14!, unfortunately the protein expression was not nearly as good (on a per cell basis) as when inducing at OD 0.5 and harvesting 2hr later with an OD of 3.5.
Hi Gary,
I didn't try it myself, but the theory says this is the way to go with toxic proteins. You can induce at higher OD values and continue incubation for about 2 h before harvesting cells. But... in the case your promoter is leaky (which isn't ulikely in TB) you risk having your cells killed already at low to medium OD values. If not, then either the toxicity is not a serious issue or you are selecting for mutants. We have seen cases with toxic proteins where only mutant strains survived.
Try auto-induction. The induction only kicks in at high O.D.s (probably close to entering or even in the stationary phase) when the original glycerol carbon source is exhausted and the cells switch to the secondary lactose carbon source.
If your protein is truly toxic then you could also try the C41 or C43 e.coli strains that can better tolerate production of some toxic proteins.
Thanks for the information, my protein is not toxic, just trying to boost yield. Would like to avoid auto-induction if possible.
I think that the only way you will obtain those ODs will be in a fermenter or bioreactor- type vessel rather than shaken flask. Otherwise it is just expanding the volumes in flasks to increase your biomass.
Hi Nick,
Yeah, that is what I thought, but the spec. sheet as well as other sources say that TB with "added" glycerol and Mg can reach 18 OD in a shaker flask at 350 rpm. I do not need 18, 15 OD at harvesting time would be nice! I did find a website that sells media that allows up to 40-50 OD in "shaker flasks" at 350 rpm, but the media is expensive ($70/L).
Hi Gary,
How much protein do you want to make? You may be able to improve yields by adding a fusion protein, switching promoters etc, rather than ever-expanding your culture volumes or spending a small fortune on media. This approach may also improve the solubility (I assume you want it soluble) and stability of your protein as well as the yields.
Hi Nick,
40-80 mg would be fine for one 3 L batch (last time got ~20 mg purified), expression is fine, soluble, easy purification, just that collaborators need a lot.
Hi Gary, did you end up improving the yield? I know it's a bit late for this now but the reason you are getting same yield even after high OD is that a fixed amount of the protein can be in inclusion body and that a fixed percentage is expressed as soluble form. Unfortunately, proteins are not always either soluble or insoluble (unless it's like a membrane protein or some weird special protein). In my case I started doing that (higher OD induction) since most of my protein goes to the inclusion bodies and I had to refold it in vitro... you might see that your fraction of inclusion bodies has indeed increased between low and high OD.
Hi Hareth,
Thanks for the information, I found that inducing at higher ODs made the purification of IBs messy, so went back to inducing around 0.5 ODs and got the normal clean IB prep.
Hi Gary, I regularly induce in TB at OD~2.5, and express until OD~10-12(pET28a, BL21). I do add additional antibiotic around doubling (to maintain selective pressure) and additional IPTG (total induction time
Thanks Ethan, so how much antibiotic (and what antibiotic, amp. versus carb.) and IPTG do you add in total for a 1L culture over the >4h time course? (assume @37oC) I was thinking I may need to add more IPTG, but things go so messy I just went back to the normal 0.5 OD induction protocol. I express proteins that are both solubile (use 6M GuHCl to lyse cells) and insoluble (IBs) which I spin out after lysing cells and then solubilize with urea then dialyze/refold.
I use Kanamycin currently, but have used Amp for various things in the past. Usually for Amp (or Carb) I would probably use a working concentration of 100ug/mL (1mL of 100mg/mL stock per L) initially, then 200ug/mL Amp (100 - 125ug/mL is likely sufficient for Carb if $$ is an issue) after doubling, since Amp degrades/gets inactivated more easily than Kan. These aren't hard set values, and will have to be optimized (I'm optimized for Kan currently at 50-75mg each time; different inhibition mechanism). IPTG I usually use 1.5-2.0 mM (~0.36 - .48 g/L) at induction (OD 2.5-3), then another 1.5 - 2.0 mM at doubling (OD ~6), and that is usually sufficient, however I've added a 3rd time in the past, but haven't properly quantified the effect. BTW, what do you mean by "messy"?
I've found that dialyzing the 8M sonicated (MUST be sonicated or mechanically lysed as well to solubilize everything) GuHCl directly is a much cleaner/higher yield prep than spinning and trying to use Urea. I usually spin out the next day after dialysis is complete. However, that may not be the case for your protein. As a caveat, these are conditions I've had a lot of success with, but as we all know, they don't apply across the board, so just some food for thought! :-)
Hi Gary,
I though a bit about this and I read your response to Ethan. What I do is that I wash The IB with a buffer (Tris50mM, Urea 2M, 500mM Nacl and TritonX1%) this will ensure that the Membrane proteins, lipids and Nucleic acids are efficiently removed from the pellet you have. You can then add another step with centrifugation/wash with the same buffer lacking Tritonx if it interferes with downstream applications (such as Ni+ columns). Thorough resuspention with those buffers is key- I use a syringe/needle-it's a couple of hours extra work but you will instantly see the difference of purity on your SDS-gels
Good luck
Thanks Ethan, I was thinking the standard IPTG amount was probably not enough. By messy, I meant that the yield of target protein versus non-target went way down.
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