Pichia pastoris question

Lee J. Lefkowitz llefkowi at checfs1
Wed Jan 18 20:06:42 EST 1995


I am trying to express a phospholipase A2 from cobra venom in Pichia pastoris
and have had some troubles.  I think that this may be due to the
toxic properties of PLA2.  Does anyone have any experience with the
lipid composition of Pichia pastoris or know of an assay to check if
the induction is working (perhaps an assay for alcohol oxidase)?

I would also like to check the integrity of the cells following induction
with methanol.  I know that it is possible to assay for an intracellular
enzyme such as lactate dehydrogenase in certain cells and therefore
determine if the cell membranes are remaining intact.  Is there a 
different protein that can be used for such purposes in Pichia?

I am hoping that the answers to these questions will tell me if the
toxic properties of PLA2 are complicating expression.  Any advice
would be much appreciated.

Lee Lefkowitz
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