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Pichia problems

Alex Beeser A_Beeser at fccc.edu
Mon Jul 9 13:33:16 EST 2001


  I am new to pichia and have a rather unsettling observation.  I'm
expressing a protein fom the pPICZalpha constructs, and it expresses in
small scale better in mut+ strains than Mut- (X-33 vs KM71H). I've done
pilot experiements with what I think are single integrants and multiple
integrants in X-33 and the multiple integrant clone seems to express
better, which is fine.  By 24 hours post induction in buffered methanol
media ( BMMY) I can see myc- X reactive bands in the cell lysate, by 48
hours I can get a X-reacrive band that would correspond to the processed
secreted form   Thinking longer is better I let the culture co for
another 24 hours and by 72 hours there is no longer a cross reactive
band from the supernatant.  My question is thus, where did the protein
go ?  Are proteases the likely culprit ?  As the medium is buffered and
pichia expresses low levels of secreted proteases anyways, I'm not sure
if this is likely.  If one can discount proteolysis, wouldn't one expect
an increase of the secreted protein as a function of time ?

  Thanks in advance.



Sandy Beeser






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