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High Yield Protein Production from Pichia pastoris Yeast: A Protocol for Benchtop Fermentation

th limiting nutrients can be pumped into the vessel at a rate that is capable of replenishing the nutrients that are depleted. Shakers can only run in a batch mode, meaning that the growth of the cells is limited by the nutrients present in the medium at the time of inoculation. The fermentors fed-batch mode further enables methanol flow rates to be controlled to condition the cells to the presence of the methanol, as well as provide methanol at the proper rate to allow addition of just enough methanol for protein synthesis while preventing excess methanol addition which can cause toxicity.

Researchers have found that optimum protein production in P. pastoris occurs at 30C, and that all protein expression ceases at 32C. However, high heat loads occur when P. pastoris is actively growing or expressing high levels of protein. In actively growing shake flask cultures, it is not uncommon for the temperature to increase 25C if left uncontrolled. Therefore, it is imperative that all aspects of the fermentor, including temperature controller, heat exchanger, vessel and piping, must be designed to regulate temperature for optimum protein production. In addition to the fermentors internal controller, an external bioprocessing software (BioCommand, New Brunswick Scientific) is routinely used to supervise the process, as well as to provide optimal nutrient feed rates, based on either the current status of the culture or to actuate pre-determined control scenarios.


Fermentation Protocol
Research was conducted in BioFlo 3000 benchtop fermentors (New Brunswick Scientific) (Figure 1) with interchangeable, autoclavable vessels of 1.25 to 10 L working volume, as well as in a BioFlo 4500 fermentors with sterilizable-in-place vessels of 15 L and 20 L working volume, (New Brunswick Scientific
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