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Dear Francisco,
My first and only experience with large scale protein expression is with Pichia
(several years ago, I must say). The kits may have changed since I did this,
but, here are some points and practical advice that I learned while I used this
system:
1. It worked very well for the membrane-bound protein that I was working with
(hMAOB and varying mutants). It was definitely worth the effort!
2. I would recommend an inducible vector if you think your protein may be toxic
to the organism. I also recommend a multi-copy vector. I saw a dramatic
increase in expression with the higher G418 resistance (you may not be using
this technique)
3. If you induce with MeOH, it is absolutely crucial to monitor cell growth to
determine when to add MeOH -- do not go by number of days that it has grown. In
my case, they always grew slower than was predicted and this affected when I
could give the first MeOH addition.
4. If you induce with MeOH, be very careful not to "overdose" the cells. There
is a definite balance that needs to be achieved here. For large scale
expression (2 L and up), I achieved my best results when I fed the MeOH using a
syringe pump (instead of the peristaltic pumps on the fermenter that I used). I
saw many more O2 fluctuations with the peristaltic pump due to its uneven flow
and I think it really affected my results.
5. Don't forget the antifoam when scaling up!! These little guys grow very
agressively and there were several mornings that I came in and had a big mess
of Pichia to clean up! I actually started adding an overflow container to avoid
these very annoying occurrances.
6. Search the journal "Protein Expression and Purification" for relevant
references -- there should be several in there.
Good luck!
Paige Vinson
Paige Vinson, PhD
Applications Scientist
Rhombix Products for Automated Crystallization
Thermo Electron Corporation
7109 Bakers Bridge Ave
Brentwood, TN 37027
ph: 1-615-263-9406
www.thermo.com
-----Original Message-----
-----Original Message-----
From: [EMAIL PROTECTED] on behalf of [EMAIL PROTECTED]
Sent: Thu 10/12/2006 2:38 PM
To: [email protected]
Cc:
Subject: [ccp4bb]: Off Topic : Pichia expression for Structural Biology
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Dear All :
Sorry for the non-CCP4 question. I am currently starting to work on
Pichia
expression for the production of human proteins for x-ray studies.
I was wondering if any of you have experience in Pichia expression for
structural studies, and would like to share some knowledge with me (I
am a
complete begginer in the field). I am interested in the expression of
human nuclear proteins, and I would like to hear opinions and feelings
about what system to use (intracellular vs secreted, constitutive vs
inducible promoters, etc.). If any of you have some good references
about
the topic I would also appreciate.
Many thanks for your help.
Cheers
Francisco
-----------------------------------------
Francisco J. Enguita, Ph.D.
Host-pathogen Interactions Group
Macromolecular Crystallography Laboratory
ITQB
EAN, Av. da República
2781-901 Oeiras
Portugal
Phone : +351-21-4469663
Fax : +351-21-4433644
E-mail : [EMAIL PROTECTED]
-----------------------------------------
