TITLE: Protein expression in Komagataella phaffii (formerly Pichia pastoris)
AUTHORS: Maja Rennig, Kristoffer Bach Falkenberg, Cristina Hernandez Rollan, Andreas Birk Bertelsen, Morten Norholm

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>The methylotrophic yeast </span><span style = "font-style:italic;">Komagataella phaffii</span><span> (formerly </span><span style = "font-style:italic;">Pichia pastoris</span><span>) 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 </span><span style = "font-style:italic;">K. phaffii.</span></div><div class = "text-block"><span>The protocol found here is modified from the protocol provided in the </span><span style = "font-style:italic;">Pichia </span><span>Expression Kit (Catalog Number K1710-01, Invitrogen).</span></div></div>

[STEPS]
?. [Prepare media and stock solutions]
Prepare BMGY and BMMY mediaIngredients (final concentrations):  yeast extract peptone mM potassium phosphate, pH 6.0 YNB  biotin  glycerol (BMGY) or  methanol (BMMY)
K. phaffii cells exhibit optimal growth with higher YNB concentrations than S. cerevisiae
?. [Prepare media and stock solutions]
Dissolve  of yeast extract,  of peptone in of water
10 g
20 g
700 mL
?. [Prepare media and stock solutions]
Autoclave for   on liquid a cycle program
?. [Prepare media and stock solutions]
Let cool down to room temperature, then add the following:  potassium phosphate buffer,   (autoclaved stock solution) YNB with ammonium sulfate without amino acids (filter sterilized stock solution) biotin (filter sterilized stock solution)
100 mL
100 mL
2 mL
?. [Prepare media and stock solutions]
For BMGY add   glycerolFor BMMY add  methanol
100 mL
100 mL
If not used directly, store the media at   for up to 2 months
?. [Prepare media and stock solutions]
Prepare   potassium phosphate buffer,
?. [Pre-culture - Day 2]
Transfer   of the saturated culture into  BMGY in a 500 mL shake flask with low baffles and grow overnight at
2.5 mL
100 mL
28 °C
It is also possible to grow the cells at
?. [Expression - Day 3]
Measure OD600 of the overnight culture
?. [Expression - Day 3]
Pellet  of the culture in a 50 mL falcon tube at
50 mL
Centrifuge: 4000 34, 10 min
?. [Inoculation - Day 1]
Inoculate  of BMGY in a 250 mL shake flask with a single colony of K. phaffii GS115 with the desired construct integrated on the genome.
25 mL
?. [Prepare media and stock solutions]
Prepare  YNB with ammonium sulfate without amino acids
?. [Prepare media and stock solutions]
Prepare  biotin solution
?. [Prepare media and stock solutions]
Combine  of   K2HPO4 and  of   KH2PO4
132 mL
868 mL
?. [Prepare media and stock solutions]
Confirm that the  and adjust using phosphoric acid or KOH
?. [Prepare media and stock solutions]
Sterilize by autoclaving for  on liquid cycle program and store at
0 Room temperature
The shelf life of this solution is greater than one year
?. [Prepare media and stock solutions]
Dissolve  of yeast nitrogen base (YNB) with ammonium sulfate and without amino acids in  of water. The solution can be heated to dissolve YNB completely in water
134 g
1 L
Alternatively, use   of YNB without ammonium sulfate and amino acids and   of ammonium sulfate.
?. [Prepare media and stock solutions]
Sterilize by filteration and store at
4 °C
The shelf life of this solution is approximately one year
?. [Prepare media and stock solutions]
Dissolve  biotin in  of water
20 mg
100 mL
?. [Prepare media and stock solutions]
Sterilize by filteration and store at
4 °C
The shelf life of this solution is approximately one year
?. [Inoculation - Day 1]
Incubate the culture  at  with 250RPM shaking until saturation
28 °C
It is also possible to grow the cells at
?. [Expression - Day 3]
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: V_{BMMY} = \frac{OD_{600,measured}\cdot V_{pre-culture}}{50}
?. [Expression - Day 3]
Grow the cells at  and 250 RPM of shaking
28 °C
It is crucial that the temperature does not exceed   when producing protein. It is advised to double-check the incubator's temperature reading with a thermometer.
?. [Feeding - Day 4]
Add 1% methanol to the expression culture ( of methanol to  of BMMY culture) around 24 hours after inoculation
1 mL
100 mL
?. [Feeding - Day 5]
Add 1% methanol to the expression culture ( of methanol to  of BMMY culture) around 24 hours after last addition of methanol
1 mL
100 mL
?. [Feeding - Day 6]
Add 1% methanol to the expression culture ( of methanol to  of BMMY culture) around 24 hours after last addition of methanol
1 mL
100 mL
?. [Harvesting - Day 7]
Harvest the cells by centrifugation at  and discard the pellets
Centrifuge: 4000 34, 10 min, 4 10
?. [Harvesting - Day 7]
Keep the sample  when working with it and at  or  for storage
on ice
4 °C
-20 °C
?. [Expression - Day 3]
Inoculate  BMMY medium with  of the normalized pre-culture, giving a start OD600 of 1.
100 mL
2 mL