Biology Reference
In-Depth Information
10-mL sample
via
the sample port for assay and determination of cell wet weight
and/or OD
600
(
see
Note 11
).
2. Adjust the impeller speed during the course of the fermentation to maintain dO
2
above 20%.
3. When dO
2
“spikes,” take a sample then add 5 mL 100% methanol (toxic) via the
inoculation port, continuing to adjust the impeller speed to maintain dO
2
above
20% (
see
Note 12
).
4. Aseptically, fill the nutrient-feed aspirator with the methanol feed solution and
connect the aspirator to the vessel via the nutrient-feed pump.
5. When dO
2
spikes for the second time, start the methanol feed at 2.5 mL/h,
increasing to 5 mL/h after 6 h. Keep dO
2
above 20% and take samples regularly.
Add 0.1-mL aliquots of antifoam solution, as necessary (
see
Note 13
).
6. To prevent methanol accumulating in the vessel, while at the same time main-
taining optimal cell growth, the feed rate of methanol can be increased with time,
but must remain limiting. Under conditions of limiting carbon source, the respi-
ration rate rises and falls as methanol is constantly depleted then replenished in
the vessel, this is signalled by oscillations in dO
2
levels (
see
Note 14
).
7. Continue the fermentation until it has consumed at least 200 mL methanol feed
solution (
see
Note 15
).
3.4. Processing Culture Supernatants
This section of the procedure has to be tailored to the particular protein of
interest, depending on its stability, its isoelectric point and any individual char-
acteristics that may be exploited for affinity purification. In addition to the
protein of interest,
P. pastoris
secretes a small number of proteins with isoelec-
tric points in the range pH 3.0-5.0 and a nonproteinaceous high-molecular-
weight species (possibly glycosaminoglycan), which is anionic under mildly
acidic or neutral conditions of pH. With this in mind, and in the absence of an
affinity purification step that can be exploited, we recommend an initial cat-
ion-exchange purification step to concentrate and partially purify the secreted
protein of interest. However, if this protein is labile or insoluble under acidic
conditions, other initial purification methods should be assessed.
1.
Pellet the cells by centrifugation at 2000
g
for 15 min. Remove the supernatant
and determine its volume.
2.
For fermentations run at pH 6.0: Wash the cells by resuspending the cell pellet in
a volume of 100 m
M
HCl equivalent to the original supernatant volume. Pellet
the cells by centrifugation at 10,000
g
for 10 min. Remove the supernatant and
pool with the original sample of supernatant (
see
Note 16
).
3.
For fermentations at pH 3.0: As for
Subheading 3.4.2.
, but wash the cells with
distilled water (
see
Note 16
).
4.
Adjust the pooled supernatants to pH 3.0. Clarify by filtration through a 0.2-
m
membrane (
see
Note 17
) and load onto a column of SP-sepharose, pre-equili-
brated with 5 column volumes of citrate equilibration buffer.
µ
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