Origami and other folding strains

Michael L. Sullivan mlsulliv at facstaff.wisc.edu
Fri Mar 16 13:51:57 EST 2001


Thanks for the input.  In answer to your final question, no I haven't
checked the few transformants for activity.  I wasn't optimitic the few I
did recover would have activity, since the numbers suggest there would be a
strong selection against it.  I suspected that the few transformants I was
recovering had probably lost the ability to express an active protein.  I
waited to try the pLysS experiment first.  Of course now I will go back and
check it out.

Mike


>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
>
>NOTICE: Incoming messages larger than
>300 kb are rejected by our mail server
>
>
>
>-----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
>
>---


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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