Origami and other folding strains

Alejandro Miguel Martin Dunn alejandro.martin at cigb.edu.cu
Fri Mar 16 12:19:31 EST 2001


Mike,

Never worked with the Origami strain, but if it really allows
intracellular disulfide bonding, and if your protein is highly toxic
when properly folded/all SS bonds formed etc., then chances are you will
never get transformants even with pLys. 
Try a different host which does not express T7 RNA pol, and deliver the
T7 RNA pol. with a lambda or M13 phage.
Is your protein expressed in the few transformants you get with your
cloned gene?

regards,
Alejandro

---------------------------------------------
Alejandro Martin
Centro de Ingenieria Genetica y Biotecnologia
PO Box 6162, C. Postal 10600
Ciudad Habana, CUBA
Tel: (53-7)216221, FAX: (53-7)214764

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-----Original Message-----
From: "Michael L. Sullivan" [mailto:mlsulliv at facstaff.wisc.edu]
Sent: Friday, March 16, 2001 11:51 AM
To: methods at hgmp.mrc.ac.uk
Subject: Origami and other folding strains


I've been trying to express the protein of my cloned gene in E. coli.
It
expresses reasonably well as inclusions and inactive soluble protein.
I've
been trying to get active protein now though.

A few weeks ago I decided to try Novagen's Origami strain, which is
supposed to allow disulfide bond formation.

When I transformed my expression construct, I found that I recoverd few
or
no colonies.  Transforming just the pET vector gives numorous colonies.
Additionally, transformation of the expression construct into another
expression host [like BL21(DE3)RIL], gives numerous colonies.

I actually found this result encouraging, reasoning that failure to
recover
colonies might be due to leaky expression of my cloned gene, which is
perhaps toxic.  Consequently, I made a strain with  pLysS, which I
happened
to have, since this should supress basal expression.  I then used this
new
strain for to transform my expression construct and pET vector control.

Well, I've gotten the same result as I did without adding pLysS:  I
recover
hudreds of colonies when I transform the emptly pET vector, but got only
one colony from the expression construct with my gene of interest.  To
be
rigourous, I suppose I really ought to check to make sure that pLysS
really
did get into the strain (I was anxious to continue and assumed that
because
it had the right marker, it most likely has pLysS).  However, assuming
it
does, I'm trying to figure out how to proceed.

I'm wondering if I should try pLysE, which produces even more lysozyme,
and
which would (supposedly) supress basal expression even more.  Another
thought I've had is that perhaps some of the other strains that allow
disulfide formation might be "less good" than Origami, and would allow
me
to recover transformants, although they might make less active protein
when
induced.

I'd be interested in input from anybody whose had experience with this.
I'd like to have some sort of plan before I start buying more plasmids
and
strains!

Thanks,

Mike Sullivan

Michael L. Sullivan, Ph.D

U.S. Dairy Forage Research Center
1925 Linden Drive West
Madison WI, 53706

(608) 264-5144 Phone
(608) 264-5147 Fax

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