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pichia vectors

Ashwini Kamath-Loeb askamath at post.its.mcw.edu
Thu Aug 25 21:09:04 EST 1994


Hi,
I wanted to share my experiences with the Pichia expression system as well.
I have been workinh with it for the last 3 months and also have had no
success in expressing my protein.  The construction of the DNA of interest
into both of the secretion vectors worked really well and I had no
problems.  Sequencing checked out fine.  I did not have problems
transforming yeast spheroplasts but the # of his+ recombinants were far
lower than the figures that Invitrogen mentioned.  Nonetheless, I had
several clones to screen on MD and MM plates.  I got 10-25% colonies that
either were no growers or poor growers on MM plates.  I took this one step
further and did yeast colony PCR to identify those that had picked up my
DNA.  Mind you not all potential candidates were positive by PCR and I don't
know why.  But I took the candidates that were positive and checked to see
their expression.  The first time I got one clone that looked encouraging
but I have not been able to reproduce it.  The controls check out ok.  I too
ran into the problem of bacterial contaimination a couple of times even in
BMGY and BMMY medium but have managed to deal with it by including a
combination of Pen/Strep to the medium.  This does not affect the growth of
Pichia nor does it interfere with protein expression with the positive
control.  But none of the condition seem to have helped my protein.  I have
assayed the supernatants and cewll pellets by a # of techniques and have not
found my protein anywhere.  I don't have any more ideas on why this system
has not worked for me but I know several people at Vanderbilt also had
problems and gave up.  One of them expressed his protein intracellulary and
had good success but he also could not get the secretion vectors to work for
him.  If anyone gets the system to work for them please post your comments
in this newsgroup.  Thanks and Good Luck
Ashwini Kamath-Loeb



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