Secretion of protein into growth medium

Michael Tyo mtyo at ultranet.com
Mon Feb 12 16:38:06 EST 1996


In article <17721A6F6S85.NATARAJ at UNIVSCVM.CSD.SCAROLINA.EDU>,
NATARAJ at UNIVSCVM.CSD.SCAROLINA.EDU wrote:

> 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

We had the same observation working with a gene whose product is
sequestered into inclusion bodies: Good expression in shake flasks, low or
no expression in a fermenter. After investigation, we realized that
glucose (which we added to our fermenter's medium) prevented the
expression (or enhanced the degradation) of our protein. Our solution was
simply to supplement LB medium with glycerol, rather than with glucose.
Try it in a shake flask first (compare LB alone, LB plus glucose, and LB
plus glycerol). Hope it works.

--Mike



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