Pichia secretion proteins

SANDY BEESER thebeez1 at prodigy.net
Wed Jul 18 18:07:19 EST 2001


""W. Stuart Annan"" <wsa at st-andrews.ac.uk> wrote in message
news:Pine.SOL.3.96.1010718104631.28704A-100000 at edge...
> Hi,
>
> Does anyone know what proteins are naturally secreted by Pichia into the
> culture media? I've read somewhere it's about 4-5 proteins but, I can't
> find any mention of what they are.

   Well one of them is definately a protease, irrespective of what
invitrogen says.  I can take a 14 microlitre
aliquot of clarified BMMY, add a little PMSF, shake it a little and then add
5 microlitres of acetylated BSA and incubate it on at 30 C.  Want to guess
what my gels look like the following day ?  I'm not sure whether this says
that it makes a non-serine protease or a whole lot of one.  To thier credit,
even if you run as much sup as i can fit in the gel, I don't see any bands
on a coomassie but there are definately proteases present.

> The reason behind my request is that I've been having trouble getting my
> protein of interest to bind to Nickle column and I'm now going to try ion
> exchange chromatography and it would be useful to know what proteins may
> also bind to the column.

   Can you see the secreted protein but just cant get it to bind ?  If your
using the pPICZalpha based expression vectors a anti-myc western might tell
you whether the C-terminal is intact enough to recognise the tag ( from
memory it goes protein of interest-myc-his6.  You could also try to find a
little of the anti-his6 antibody from someone to test the sup directly
 this should work irrespective of the choice of vector).  I realise that
this isn't likely to be of much help, but if you do find out what the
secreted proteins are I would appreciate it if you would post it.


Sandy





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