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Pichia vectors

Ashwini Kamath-Loeb askamath at post.its.mcw.edu
Thu Aug 25 21:15:24 EST 1994


Ashwini Kamath-Loeb (askamath at post.its.mcw.edu) wrote:
: 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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