This method can help in the pre selection of chromosomes clones by Pichia pastoris strains expressing recombinant proteins under de-repressed conditions by a simple shake flask cultivations prior to up scaling. The main advantage of this technique is that high level methanol free protein expression can be achieved using Pichia pastoris, which is supported by the exact online monitoring of important cultivation parameters. Generally, individuals new to this method may struggle because the time point for starting the glass grow feed has to be adapted to the growth characteristics of the specific production strain.

Before setting up the main culture, seed a single fresh colony of the expression strain of interest into 5 ml of yeast pepto and dextrose broth in a 50 ml conical tube. Leave the lid slightly open to allow proper aeration. Than, place the culture in a shaker at 28 degrees Celsius, 80%humidity and 100 to 130 RPM overnight.

The next morning start the software for connecting the devices via Bluetooth. Next, press the silver button on the front of the biomass measuring device to turn on the Bluetooth connection to the device and click find devices in the computer monitoring software. Drag and drop the found devices to the squares in the measurement tray and click connect.

When a successful connection is made, a control screen will appear. To set up an experiment, click start measurement. Name the experiment and check if all parameters required for the monitoring are enabled.

Set the interval to three minutes and the average measurement points to 11. Enter the names for the samples and skip the angle calibration. Measure the cell density of the overnight culture diluted at a one to twenty ratio in cultivation medium in a spectrophotometer at a 600 nanometer wavelength.

Inoculate 50 ml of the medium with the overnight culture to an OD 600 of 05. Than, place the flasks in the detectors at 28 degrees Celsius, 130 RPM and 80%humidity for five minutes. Click start measurement in the software.

Four hours after the inoculation, use a 2 ml sample for cell density measurement as just demonstrated. When the oxygen concentration is approaching zero and the cells are on the exponential growth phase add four glycerol feed disks to each flask under sterile conditions. Than, return the flasks to the shaking incubator for an additional 60 to 90 hours.

Take samples every 24 hours to monitor the cell density and the protein expression by the assay of choice. At the end of the cultivation, transfer the cultures into 50 ml conical tubes for centrifugation. Export the online measurement data as a spreadsheet file from the software for further analysis.

In this representative cultivation, the starting glycerol concentration was 5%Although the feed disks were added when the biomass was increasing exponentially, the oxygen decreased to nearly zero. Lower glycerol concentrations reduced the effect of the short term oxygen limitations and are therefore recommended for de-repressed protein expression. In this representative experiment in which human growth hormone was expressed under the control of a carbon source repressed promoter upon glycerol depletion the protein expression by de-repression was ensured by the addition of four feed disks per flask.

As expected the human growth hormone expression and the biomass development increased over time in the presence of the glycerol feed disks, while the protein expression decreased over time and the biomass remained constant in absence of the glycerol supplement. While attempting this procedure, it's important to remember to carefully observe the cultivation parameters to determine an optimal expression start time. Following this procedure other methods like protein concentration determination western blot analysis or activity assays can be performed for protein yield evaluation as well as small scale bioreactor experiments to answer additional questions about the performance of the individual strain on a larger scale.

After its development, this technique paved the way for researchers in the field of methanol free recombinant protein expression to explore a promising alternative to the commonly used methanol dependent AOX1 promoter on Pichia pastoris.