Origami and other folding strains

Michael L. Sullivan mlsulliv at facstaff.wisc.edu
Fri Mar 16 11:50:59 EST 2001


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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