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.
>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
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>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.
>expresses reasonably well as inclusions and inactive soluble protein.
>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
>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
>colonies might be due to leaky expression of my cloned gene, which is
>perhaps toxic. Consequently, I made a strain with pLysS, which I
>to have, since this should supress basal expression. I then used this
>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
>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
>rigourous, I suppose I really ought to check to make sure that pLysS
>did get into the strain (I was anxious to continue and assumed that
>it had the right marker, it most likely has pLysS). However, assuming
>does, I'm trying to figure out how to proceed.
>I'm wondering if I should try pLysE, which produces even more lysozyme,
>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
>to recover transformants, although they might make less active protein
>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
>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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