T7 promoters and expression
jwoodget at oci.utoronto.ca
Tue Jun 7 19:55:56 EST 1994
In article <199406072215.PAA13126 at net.bio.net>, LKM100F at ODUVM.CC.ODU.EDU
("Laura K. Moen") writes:
> Has anyone else experienced such difficulties with T7
> promoters? I am surprised, but I am not exactly the most experienced
> individual with protein expression. Does anyone have any suggestions for
> ways to suppress the background expression in our uninduced cells?
> We have tried using JM109 and XL-1 Blue cells - similar problems in
> either line.
Have you tried pLysS (or pLysE) cells as originally described by Studier et
al.? These bacteria express lysozyme on a chloramphenicol selectable plasmid
and have T7 polymerase intergated into the chromosome under an IPTG inducible
promoter. Lysozyme inhibits the basal levels of T7 polymerase thus cutting
down the background. These cells have the added advantage of being easily
lysed by 3X freeze/thaw cycling which releases some lysozyme which then eats
the cell wall.
I'm sure they're commercially available (but don't remember where from).
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