Pichia expression

Clifford Beall beall.3 at OSU.EDU
Mon Nov 7 10:28:18 EST 1994


In article <94308.1439123LCID at QUCDN.QueensU.CA>, <3LCID at QUCDN.QueensU.CA> wrote:

>I am about to try expressing my protein using the Invitrogen Pichia Pastoris ex
>pression system.  I am using the pPIC9 vector which excretes the protein using
>the alpha factor signal sequence.  My questionis : has anyone tried messing wi
>th the sequence between the signal cleavage site and the first restriction site
> SnaBI.  The manual advises not changing this sequence but when I asked Invitro
>gen they said they hadn't tried changing it.  Is the EAEAY after the cleavage s
>ite necessary for efficient cleavage? I would like to eliminate as much of this
> sequence as possible so that little or no extra sequence is tagged onto the en
>d of my protein.  i would appreciate any suggestions or info on this vector spe
>ciffically or alpha factor cleavage in general.
> Thanks
> Carl DeLuca

Dear Carl,

I did a construct that had only the first glutamate after the cleavage
site, followed by my sequence (the second alanine is coincidentally the
same) and it was cleaved and secreted at reasonably high levels.  I didn't
characterize the N-terminus, but the band was the right size on SDS-PAGE. 
In general, I think it is a good idea to try an unknown with both the
pPIC9 and the pHIL S1 (pho 1) signals simultaneously, as there is no good
way to predict what will work.  It takes a fair amount of time to get
transformants, so you don't want to start again from scratch if you have
problems.

All the best,

Cliff Beall
Research Scientist
Neurobiotechnology
Ohio State University



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