Yeast Spheroplast Protocols

whitman at arris.com whitman at arris.com
Wed Jun 29 12:27:12 EST 1994


In article <9406270806.AA29932 at cscgpo.anu.edu.au> Klaus.Matthaei at ANU.EDU.AU
writes:
>
>27-6-94
>
>Dear Netters,
>        I am writing on behalf of a colleague who has turned his hand to
>protein expression using the Pichia Yeast System from Invitrogen. In
>particular the query relates to formation of spheroplasts and subseqent
>transformation with the recombinant plasmids.
>        He has tried the "standard" protocol(in the Invitrogen Pichia
>manual), for spheroplast formation, of sorbitol washes, Zymolase treatment
>then PEG/CaT transformation.This takes a long time (about 5-6 hrs, ends up
>a long day etc., etc., etc....) and he was wondering if there are any
>shortcuts or even shorter protocols. Or is it a case of practice makes
>perfect?


One short cut I have used is to discontinue the first part of testing for
percent spheroplasting, and just using 15-16 minutes for Zymolase treatment. 
When I spoke with Invitrogen they claimed to have times usually ranging from
12-18 minutes, and from my own first trials with this procedure I got 14-16
minutes.  After deciding to forgo this portion of the procedure I have still
had good transformation results.  This easily cuts out two hours of work,
turning your very long day into just a normal long day.


>        Also, which is the preferred method for spheroplast transformation:
>PEG/CaT or Lithium Chloride or Electroporation?
>        BIO101 seem to have a kit which makes spheroplasts in 1 hour. Has
>anyone had experience with it or is it just pie-in-the-sky?


Unfortunately the Bio101 kit is only good for the yeast strain they provide
with the kit, not for Pichia.

In addition, I have plans for the next time I need to do a transformation to
use Invitrogen's electroporation procedure(located at the back of the manual)
at the same time as the spheroplasting procedure in order to do a side-by-side
comparison.  Based on their materials, the electroporation should work well. 
However you lose the possibility for multiple integrations, and thus may get
strains with low levels of protein production.  The same problem is true for
the Lithium chloride method.

>stuff deleted

Good luck!  If you come up with anything else, please let me know.

Laura Savel Whitman
Molecular Biology
whitman at arris.com





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