In theory, the upscaling of culture volume should not affect protein yield per ml of culture. In practice, the opposite happens pretty often. Aeration of the large culture volumes in shaking flasks is usually less efficient, and the warming-up to 37oC is slower. Your protein's expression unit or protein itself may be sensitive to such changes and either not synthesized in the first place or synthesized and quickly degraded / aggregated. You may try to lower the volume of growth medium per flask and pre-warming the medium to 37oC prior inoculation. Another possible explanation is that when upscaling the volume you unknowingly change composition of the growth medium. It happens when you use the same stock of autoclaved medium for small-scale experiments but prepare the medium anew for each large scale growth. Same goes for inducer if you use one. You may prepare a large volume of the medium but first test the batch in a small scale protein isolation experiment. To save time and labor, you may run crude lysate on SDS-PAGE if your protein's band does not overlap with others.

Another possibility is the loss or inactivation of protein expression unit on the plasmid if the protein is somehow toxic to the cell. If you start your small scale experiment from a single colony and large scale growth from a starting culture obtained from a single colony, in the second case the culture may have enough time to be taken over by a mutant, in which your recombinant protein does not express. Just because the large culture has to pass through two growths instead of one. You may keep an aliquot of the starting culture and process it alongside with an aliquot from the upscaled culture to see if your protein is present in the starting culture and absent in the upscaled culture.

Dear Mallar Dasgupta

It is something quite frequent that protein productivity in the scale up may decrease.

In my experience this may be due:

- Lower oxigenation of the bacterial cells in the scale up:

e.g Did you maintain the same ratio between shake flask volume and colture media volume? Eg. 50ml in 250 ml flasks and 500 ml in 2,5 liter flask? What about shaking rate?

- Different temperature regulation: For example if you are growing the cells at 37°C and you shift down the temperature at 25°C or 17°C just before the induction, the drop down of the temperature inside the culture is slower and it may impact the solubility of the role.

- Low efficiency of the cell lysis using higher pellets;

For example with higher biomass the sonication cicles should be optimized

best regards

Manuele

Greetings Dr. Stepanov and Dr. Martinelli, your suggestions helped me immensely in optimizing the protocol. First of all to mention, I generally use 16 degrees (18 hours) incubation period after addition of 0.05 mM IPTG since the size of the protein is quite high. However, the main problem which I came to know was quite similar to what Dr. Stepanov suggested. The protein was indeed toxic to the bacterial cells. In small scale, I usually used to induce at O.D of 0.20 but still observed the protein in eluted fractions since the toxicity over smaller volume was probably limited and tolerable. However, replicating it at large scale caused issue. At OD of 0.2, not much concentration of cells were there and the induced protein started killing those few cells and somehow resulted in repressing its own expression. The trick worked when I induced the protein at OD 0.6 and 0.9 at large scale, when already a huge no of cells have been formed and this resulted in the protein being purified.

Sounds great, good luck with your project!