Oct 27, 2020

Public workspaceProtein expression in Komagataella phaffii (formerly Pichia pastoris)

DOI
dx.doi.org/10.17504/protocols.io.bd32i8qe
  • 1Technical University of Denmark
Maja Rennig
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DOI: dx.doi.org/10.17504/protocols.io.bd32i8qe
Protocol Citation: Maja Rennig, Kristoffer Bach Falkenberg, Cristina Hernandez Rollan, Andreas Birk Bertelsen, Morten Norholm 2020. Protein expression in Komagataella phaffii (formerly Pichia pastoris). protocols.io https://dx.doi.org/10.17504/protocols.io.bd32i8qe
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 23, 2020
Last Modified: October 28, 2020
Protocol Integer ID: 34650
Keywords: LyGo, LPMO, K. phaffii, P. pastoris, protein production,
Abstract
The methylotrophic yeast Komagataella phaffii (formerly Pichia pastoris) is the most commonly used yeast species in the production of recombinant proteins. This is most likely due to its ability to grow to high cell density, to express recombinant genes in a tightly controlled manner and to efficiently secrete proteins. Despite its biotechnological importance and wide use in industry, relatively few genetic tools are readily available for academic research. Here, we present a protocol for production of proteins in K. phaffii.

The protocol found here is modified from the protocol provided in the Pichia Expression Kit (Catalog Number K1710-01, Invitrogen).
Guidelines
Transformation and integration into K. phaffii GS115 can be done utilizing the Pichia EasyComp Kit (Catalog no. K1730-01, Invitrogen).

Recipes for all media and solutions can also be found in the manual for the Pichia Expression Kit (Catalog Number K1710-01, Invitrogen).
Materials
MATERIALS
ReagentYeast ExtractCatalog #Y1625
ReagentGS115, Pichia pastoris Yeast StrainThermo FisherCatalog #C18100
ReagentYNB without Amino AcidsMerck MilliporeSigma (Sigma-Aldrich)Catalog #Y0626-1KG
ReagentBiotinMerck MilliporeSigma (Sigma-Aldrich)Catalog #B4639-1G
ReagentPeptone from soybean enzymatic digestMerck MilliporeSigma (Sigma-Aldrich)Catalog #70178-500G
ReagentPotassium phosphate monobasicMerck MilliporeSigma (Sigma-Aldrich)Catalog #P0662-1KG
ReagentPotassium phosphate dibasicMerck MilliporeSigma (Sigma-Aldrich)Catalog #P3786-1KG
ReagentMethanolMerck MilliporeSigma (Sigma-Aldrich)Catalog #34860-2.5L-M
ReagentGlycerolMerck MilliporeSigma (Sigma-Aldrich)Catalog #15523-1L-M
Safety warnings
This protocol describes the use of GMO classified organisms. Make sure that the local GMO and safety legislations are respected.

This protocol involves the use of 100 % methanol. Make sure to store and handle 100 % methanol accordingly.
Before start
Make sure to have your expression strain streaked on an YPD agar plate
Prepare media and stock solutions
Prepare media and stock solutions
Prepare Concentration1 Molarity (M) potassium phosphate buffer, Ph6

Combine Amount132 mL of Concentration1 Molarity (M) K2HPO4 and Amount868 mL of Concentration1 Molarity (M) KH2PO4

Confirm that the Ph6.0 and adjust using phosphoric acid or KOH

Sterilize by autoclaving for Duration00:20:00 on liquid cycle program and store at TemperatureRoom temperature

Note
The shelf life of this solution is greater than one year

Prepare Concentration13.4 Mass / % volume YNB with ammonium sulfate without amino acids

Dissolve Amount134 g of yeast nitrogen base (YNB) with ammonium sulfate and without amino acids in Amount1.000 L of water.
The solution can be heated to dissolve YNB completely in water

Note
Alternatively, use Amount34 g of YNB without ammonium sulfate and amino acids and Amount100 g of ammonium sulfate.

Sterilize by filteration and store at Temperature4 °C

Note
The shelf life of this solution is approximately one year

Prepare Concentration0.2 Mass / % volume biotin solution
Dissolve Amount20 mg biotin in Amount100 mL of water

Sterilize by filteration and store at Temperature4 °C

Note
The shelf life of this solution is approximately one year

Prepare BMGY and BMMY media

Ingredients (final concentrations):
Concentration1 Mass / % volume yeast extract
Concentration2 Mass / % volume peptone
Concentration100 millimolar (mM) mM potassium phosphate, pH 6.0
Concentration1.34 Mass / % volume YNB
Concentration0.0004 Mass / % volume biotin
Concentration1 % volume glycerol (BMGY) or Concentration0.5 % volume methanol (BMMY)

Note
K. phaffii cells exhibit optimal growth with higher YNB concentrations than S. cerevisiae

Dissolve Amount10 g of yeast extract, Amount20 g of peptone in Amount700 mL of water

Autoclave for Duration00:20:00 on liquid a cycle program

Let cool down to room temperature, then add the following:
Amount100 mL Concentration1 Molarity (M) potassium phosphate buffer, Ph6 (autoclaved stock solution)
Amount100 mL Concentration13.4 Mass / % volume YNB with ammonium sulfate without amino acids (filter sterilized stock solution)
Amount2 mL Concentration0.02 Mass / % volume biotin (filter sterilized stock solution)

For BMGY add Amount100 mL Concentration10 % volume glycerol
For BMMY add Amount100 mL Concentration5 % volume methanol
Note
If not used directly, store the media at Temperature4 °C for up to 2 months


Inoculation - Day 1
Inoculation - Day 1
Inoculate Amount25 mL of BMGY in a 250 mL shake flask with a single colony of K. phaffii GS115 with the desired construct integrated on the genome.



Incubate the culture at Temperature28 °C with 250RPM shaking until saturation
Note
It is also possible to grow the cells atTemperature30 °C

Pre-culture - Day 2
Pre-culture - Day 2
Transfer Amount2.5 mL of the saturated culture into Amount100 mL BMGY in a 500 mL shake flask with low baffles and grow overnight at Temperature28 °C

Note
It is also possible to grow the cells atTemperature30 °C


Expression - Day 3
Expression - Day 3
Measure OD600 of the overnight culture
Pellet Amount50 mL of the culture in a 50 mL falcon tube at Centrifigation4000 x g, Room temperature, 00:10:00

Discard the supernatant and resuspend the pellet in BMMY medium to a final OD600 of 50. The appropriate amount of media can be calculated as:

Inoculate Amount100 mL BMMY medium with Amount2 mL of the normalized pre-culture, giving a start OD600 of 1.

Grow the cells at Temperature28 °C and 250 RPM of shaking
Note
It is crucial that the temperature does not exceed Temperature28 °C when producing protein. It is advised to double-check the incubator's temperature reading with a thermometer.

Feeding - Day 4
Feeding - Day 4
Add 1% methanol to the expression culture (Amount1 mL of Concentration100 % volume methanol to Amount100 mL of BMMY culture) around 24 hours after inoculation
Feeding - Day 5
Feeding - Day 5
Add 1% methanol to the expression culture (Amount1 mL of Concentration100 % volume methanol to Amount100 mL of BMMY culture) around 24 hours after last addition of methanol
Feeding - Day 6
Feeding - Day 6
Add 1% methanol to the expression culture (Amount1 mL of Concentration100 % volume methanol to Amount100 mL of BMMY culture) around 24 hours after last addition of methanol
Harvesting - Day 7
Harvesting - Day 7
Harvest the cells by centrifugation at Centrifigation4000 x g, 4°C, 00:10:00 and discard the pellets
Keep the sample TemperatureOn ice when working with it and at Temperature4 °C or Temperature-20 °C for storage