Secretion of protein int

Mary Weiss weiss at dnax.org
Thu Feb 8 12:05:10 EST 1996


         Reply to:   RE>Secretion of protein into growth medium

Natarajan,

I have also run into this problem about a year ago.  Are you using the pSW1
plasmid to produce the "leakiness" and if so did you remember to use
tetracycline in your media.  We also found different levels of induction
depending on the level of mitomycin C that we used.  Lower levels worked
better and higher levels tended to lyse the cells all together.  You also need
to look at expression of your product prior to and during induction-preferably
using something sensitive like a western blot.  I found that once in the
fermentor my product level didn't increase in the medium after the first hour
of induction and that a lot of the material was in the periplasm as well as in
the cell(inclusion body formation).  I didn't play around with changing
fermentor conditions to try and improve the secretion but probably would have
tried lowering temperature or dissolved oxygen levels.

I opted for using osmotic shock(a few different types) to remove the product
from the periplasm and that seemed to work pretty well.  It also is a lot
easier to process a small amount of shock material to put over a column rather
than 15L of media containing product.

I hope this helps.

Mary

--------------------------------------
Date: 2/2/96 11:04 AM
To: Mary Weiss
From: NATARAJ at UNIVSCVM.CSD.SCAROLINA
Hi,
   I am working with a bacterial gene which is secreted into the periplasmic
space.  I cloned it under the control of T7 promoter and in expression studies
in BL21DE3pLys grown in shake flasks, I found that the protein was secreted
into the growth medium at very high levels(>100mg/liter).  I did the
experiment
in a fermentor and the results are very different.  It is not secreted into
the
medium!  My question is what is so different in a fermentor to cause this
change in secretion.  I tried to reduce the dissolved air level in the
in the fermentor.  This did not improve the situation.  What other parameters
could be changed to get the protein secreted into the medium?  BTW the protein
IS getting secreted into the periplasmic space.  Thanks in advance for your
help.
     Natarajan


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To: microbio at net.bio.net
From: NATARAJ at UNIVSCVM.CSD.SCAROLINA.EDU
Subject: Secretion of protein into growth medium
Date: Fri, 02 Feb 96 11:52:22 EST
Message-ID: <17721A6F6S85.NATARAJ at UNIVSCVM.CSD.SCAROLINA.EDU>
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