We have attempted to express large quantities (1-100 mg) of several
proteins in Pichia to use in animal studies and for crystallographic
analysis. Specifically, we have used the Zeocin resistant vector pPICZaC
from Invitrogen, designed for protein expression and secretion. We have
succeeded in transforming the yeast with the vector containing construct
DNA, though at considerably lower yield than expected from the
manufacturer's literature.
Since then, we have screened all clones for protein expression by induction
per Invitrogen specifications (using 0.5%, 1%, 1.5% and 2% MeOH in minimal
media), sampling media and cells during a time course of 72 - 168 hours.
We did not detect significant levels of the desired protein in either the
conditioned media concentrates or cell extracts.
We are now trying to locate individuals in the scientific community who
have extensive experience in the field of large scale mammalian protein
production using yeast expression systems. If anyone on the list can help
us troubleshoot this system or offer advice regarding alternative Pichia
expression systems, their help would be greatly appreciated.
Please reply to:
breckend at pop.nci.nih.gov
Diane Breckenridge
LCMB/NCI/NIH
Bldg. 37/1E24
37 Convent Dr.
Bethesda, MD 20892
