Biology Reference
In-Depth Information
5.
Wash the SP-sepharose with citrate wash buffer, monitoring A
280
of the eluate,
until a flat baseline is obtained (
see
Note 18
).
6.
Elute bound proteins by washing the SP-sepharose with a linear gradient of
0-100% citrate elution buffer over 5 column volumes, collecting fractions, and
monitoring A
280
(
see
Note 18
).
4. Notes
1
.
P. pastoris
growth media commonly contain glycerol instead of glucose as the
carbon source. Glucose is merely chosen because of the cost and availability of
its
13
C isotopically labeled analog.
2.
Selection of the growth and induction pH of
P. pastoris
can help to overcome
proteolysis and insolubility of the recombinant product. The optimum value
should be determined in small-scale cultures prior to fermentation. Final pH
adjustment is performed in the vessel because precipitation of inorganic salts
during the sterilization process can occur if the pH of BSM >3.5.
3.
Ammonium sulphate and D-glucose can be sterilized by autoclaving. Filter ster-
ilization is used instead as a precaution against accidental losses of the isotopi-
cally labeled compounds.
4.
Different molecular-weight preparations of polypropylene glycol are available;
we routinely use 1025. The disadvantage of polypropylene glycol as an antifoam
reagent is that its presence, even at low concentrations, can significantly reduce
flow rates through certain ultrafiltration membranes.
5.
The volume of seed culture can be changed according to the scale of fermenta-
tion. Using 10% of the fermentation volume as the seed culture works well.
6.
The performance of gel-filled autoclavable pH probes will deteriorate with
repeated sterilization, but can be prolonged by periodic cleaning as recommended
by the manufacturer. Probes should be stored in 3
M
KCl.
7.
We have only used polarographic dissolved oxygen probes; information on the
use of galvanic oxygen probes should be obtained from the manufacturer.
Polarographic oxygen probes require routine maintenance to prolong their work-
ing life and these details should also be obtained from the manufacturer. Probes
should be stored wet.
8.
Facilities for temperature control vary; our vessel has an external heater and an
internal cold finger, other glass vessels may have a water jacket connected to an
external thermostatically controlled bath.
9.
As the vessel reaches its target pH and particularly on addition of PTM
1
salts,
some inorganic salts will precipitate. This does not affect cell growth.
10.
This operation will depend on the design of the controller.
11.
Regular sampling is important to monitor the growth of the fermentation, allows
assay for any recombinant product, and permits microscopic inspection of the
culture to screen for contaminating microorganisms. The precipitated inorganic
salts in the sample can interfere with these tests, but can be dissolved by the
addition of an equal volume of 100 m
M
HCl without leading to cell lysis. If
the recombinant product is acid labile, then a sample of supernatant should be
removed for assay before addition of the HCl.
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