p. pastoris protein secretion problem

Stefanie Wedepohl stefanie.wedepohl at gmx.de
Fri Oct 24 08:17:00 EST 2003


hi experts

I want to use p.pastoris for heterologous protein production. but all
attempts failed. i use invitrogen's expression kit and tried 4
different vectors, two strains, 8 constructs and 3 fusion tags. the
problem is, that somestimes the yeast produces my Protein well, but
does not secrete it into the medium. (i sequenced most of the
constructs to verify that the secretion signal is in frame) i tried
pho1, alpha-factor, native leader sequence etc. ...)  so it seems,
that my protein will not be secreted by pichia. but to give up trying,
i'd like to see a note or something in a database that tells me, why
it is not secreted. my question: is there somewhere a list or
something or a common rule for predicting secretion or intracellular
expression of a protein? something like: if your protein has sequence
GKIONVOR, than it will be retained. #

can anybody help me and explain, when a protein is retained? has
someone same experience and solved the problem ? give me a hint.

Thanx. (P.S. it is essential for me to have the protein in the medium,
so don't recommend to purify it from cell-lysates. oh by the way, a
crude fractionation revealed, that it does not stick on the outside
but is in the membrane fraction)

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