Check the surface area of the flask you are growing the cells in.  When growing the small volume you may have a larger surface area/volume than when you scale it up. 

Thanks for pointing that out, Barbara, I forgot to mention that.

We have surface/volume ratios of ~0.56 and ~0.54 for 200 and 5 mL, respectively. Ratios were measured taking into account the elliptical surface of a glass tube at 45°; The bottle stands upright and we assume it is a circle.

Of course, this calculations are made while the samples are not shaking. Estimating the area while shaking is quite tricky, though I would expect a slightly bigger increase of the ratio from the glass tube, when compared with the bottle.

Would this make such a big difference? Would you attribute this effect to oxygenation, then?

Thanks again.

Some thoughts:

1 - E.coli is one of the fastest growing cells and even at low cell concentration you need to supply oxygen as much as possible, for this purpose you can increase the shaker speed, 300 rpm - typical, use the baffled flasks, typical volume ratio is 1/10 that means 100mL medium in 1000ml EF (better 1/20 ratio =50/1000), when you increase rpm or change the volume ratio you should always check visually that the medium has rotation with high level of vortexing because sometimes even if you increase the rpm the medium does not rotate because the agitation speed is too high and the medium just stay in the middle of your flask

2 - For some reason your target protein might be expressed in IB-form when you change the volume, therefore you should check it trying the assays for IB-proteins

Thanks for the reply, Anatoliy, and for pointing out that the protein might be trapped in inclusion bodies when scaling up with the volume. It never happened before that we would get such a sharp difference, though it is definitely worth trying.

Hi Francesco,

I've had such an experience once, and it was indeed in inclusion bodies. So I had to use Urea extraction (8 M) and dialisis before being able to use the purified protein. I did not search further to try to understand why it had happened, though.

Best of luck!

Thanks for your comment, One!

We have tested 50 and 100 mL and realised we start seeing significant amount of protein in the cell pellet after lysis of the 100 mL cultures. Indeed we might have accumulation in inclusion bodies while scaling up to ~100 mL. The reason why this happens remains still unclear to me, though.

Concerning the urea extraction, my major problem is that denaturing and renaturing will result in the release of the cofactor from my protein of interest and, thus, loss of catalytic activity.

Hi Francesco,

in our Lab we work with IPTG induced proteins. We start with 3 mL to be sure that the system works and later we do the same with 5 L of culture media.

I sum up our steps:

- cloning the cDNA of interest in the expression vector pGEX-4T-1 (that will produce a fusion protein GST-your_protein)

- transform in E. coli BL21

- miniproduction (3mL) to synthesize GST-protein (induced by IPTG)

- production (5L) to synthesize GST-protein (induced by IPTG)

- purification: centrifugation to recover cells + sonication of cells + incubation of total extract proteins with Glutathione Sepharose (the GST-proteins will be bind to the sepharose) + wash of sepharose (only GST-proteins will remain in your batch) + thrombin cleavage (to eliminate GST and recover your protein in the supernatant)

I hope my short explanation can help you. If you need more information or the protocol we use, you can see it in one of our papers (http://pubs.rsc.org/en/content/articlepdf/2014/mt/c3mt00266g) or tell me and I give you the protocol we use.

Good luck!!

Anna

Have you tried to disrup your cells with Threalose in the lysis buffer? There is a theory about IB formation reported by Leibly

PLoS One. 2012;7(12):e52482. doi: 10.1371/journal.pone.0052482. Epub 2012 Dec 20. Stabilizing additives added during cell lysis aid in the solubilization of recombinant proteins. Leibly DJ1, Nguyen TN, Kao LT, Hewitt SN, Barrett LK, Van Voorhis WC.

Hi Francesco,

We have also encountered similar situations (2 different proteins) when expressing proteins using E. coli grown in deuterated minimal media. Interestingly, we did not observe any difference between small- (~ 5 mL) and large-scale (1 L) fermentation in rich media, e.g. LB. May I ask what was the composition of your growth medium?

I might add that among the ~20 proteins our lab has worked on, only the two proteins my small workgroup has been handling showed this peculiar phenomenon.

Cheers,

Chung-ke

Thanks a lot for the publication, Luis. I had a read and will give it a try soon with some of the compounds listed there.

Hi Chung-ke, we use a standard LB-Miller broth (10 g/L tryptone, 5 g/L yeast extract, 10 g/L sodium chloride).