Biology Reference
In-Depth Information
3. Methods
Media for the seed culture and the fermentation are defined such that
replacement of the ammonium sulphate, glucose, and methanol, as appropri-
ate, with their isotopically labeled analogs is sufficient to generate isotopically
labeled recombinant material. Typical routes used to generate recombinant
strains of
P. pastoris
can generate Mut
+
or Mut
s
(methanol utilization positive
or methanol utilization slow) phenotypes; the procedures described here per-
tain to strains with the Mut
+
phenotype.
3.1. Seed Culture
1.
Thaw an aliquot of a
P. pastoris
strain that has been stored as a 20% (v/v) glyc-
erol stock at -85
°
C.
2.
Inoculate a 250 mL baffled flask containing 50 mL of BMGc with 1 mL of the
glycerol stock and grow shaking at 30
C, for 16-24 h, until OD
600
= 2-6. This
will constitute the seed culture for a 0.5 l fermentation (
see
Note 5
).
°
3.2. Preparation of the Vessel
1. Calibrate the pH probe, rinse it with deionized water and introduce it into one of
the vessel's ports (
see
Note 6
).
2. Test the dO
2
probe with zeroing gel, rinse it with deionized water, and introduce
it into one of the vessel's ports (
see
Note 7
).
3. Mask the external ends of all ports and lines with aluminium foil and autoclave
tape, clamp any lines that will be in contact with the media (e.g., the air inlet and
sampling line) and sterilize the vessel containing 500 mL of BSM.
4. Sterilize 3 empty aspirators for acid and base control and for nutrient feed.
5. Aseptically, fill the acid and base control aspirators with acid and base control
solutions, respectively.
6. Connect the chart recorder and pH, dO
2
, and temperature probes to the controller.
Connect the temperature control facility (
see
Note 8
).
7. Set the impeller speed to 150 rpm and introduce 2.5 mL D-glucose solution,
10 mL ammonium sulphate solution, 2.5 mL biotin solution, 2.5 mL PTM
1
, and
0.1 mL antifoam solution via the inoculation port (
see
Note 9
).
8.
Aseptically, connect the aspirators for acid and base control to the vessel via the
acid and base control pumps and allow the system to equilibrate to the desired
temperature and pH (
see
Note 9
).
9.
Set the output from the oxygen probe to zero. Zero the chart recorder (
see
Note 10
).
10.
Connect the sterile airline, open the air outlet, and increase the impeller speed
and air-flow rates to the maximum value to be used during the fermentation,
typically 1000 rpm and 5 vol of air per volume of culture per min (vvm). After
10 min, calibrate the dO
2
probe at 100% and set the chart recorder to full scale
deflection.
3.3. Fermentation of P. pastoris
1.
Run the vessel initially at 2 vvm and at an impeller speed of 250 rpm, inoculate
with the seed culture and start the chart recorder to monitor dO
2
levels. Take a
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