Biology Reference
In-Depth Information
12. When a culture is growing normally and consumes all of the carbon source, res-
piration will slow quite dramatically causing the dO 2 level to rise quite sharply or
“spike.” Addition of more carbon source should lead to an almost instantaneous
rise in respiration signaled by a concomitant fall in dO 2 . Many fermentation con-
trollers have automated feedback control, which adjusts the impeller speed to
maintain the dO 2 at a preset level. This is very useful when the system is left
unattended for long periods of time, but can mask dO 2 spikes.
13. The addition of antifoam is usually necessary, but when growing P. pastoris at
relatively low cell densities only small quantities are required. If the methanol
feed is continued for longer periods, more antifoam solution may be needed.
Alternatively, if available, antifoam feedback control may be used.
14.
A useful guide to whether the methanol is limiting is to switch off the methanol
feed and measure the time taken for dO 2 to rise by 10%. If this time is <1 min, the
carbon source is limiting.
15.
The actual amount of methanol feed solution required to generate sufficient
recombinant material must be determined empirically. We have never used less
than 200 mL 10% (v/v) methanol.
16.
The purpose of washing the cells is threefold: i) to increase the yield of recombi-
nant protein; ii) to dilute the supernatant and therefore to reduce its ionic strength
prior to ion-exchange chromatography; and iii) to reduce the pH to ~3.0 for cat-
ion exchange.
17.
We use an Amicon hollow-fiber cartridge with an exclusion limit of 0.2
µ
m to
clarify the supernatant, but any low protein-binding 0.2-
m membrane filtration
device could be used. Alternatively, centrifugation for 20 min at > 20,000 g could
be used.
µ
18.
Washing the SP-sepharose at pH 5 should remove most of the nonrecombinant P.
pastoris proteins from the column, but may also remove the recombinant protein
if its p I <6. If this occurs, washing and eluting at a slightly lower pH may be an
option.
Acknowledgments
The authors thank the Wellcome Trust for financial support.
References
1. Bork, P., Downing, A. K., Kieffer, B., and Campbell, I. D. (1996) Structure and
distribution of modules in extracellular proteins. Quart. Rev. Biophys. 29, 119-167.
2. Baron, M., Norman, D. G., and Campbell, I. D. (1991) Protein modules TIBS
16, 13-17.
3. Hynes, R. O. (1990) Fibronectins, (Rich, A., ed.), Springer-Verlag, Berlin.
4. Mosher, D. F. (1993) Assembly of fibronectin into extracellular matrix. Curr.
Opin. Struct. Biol. 3, 214-222.
5.
Petersen, T. E., Thørgersen, H. C., Skorstengaard, K., Vibe-Pedersen, K., Sahl, P.,
Sottrup-Jensen, L., and Magnusson, S. (1983) Partial primary structure of bovine
plasma fibronectin: three types of internal homology. Proc. Nat. Acad. Sci. USA
80, 137-141.
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