protein expression in BL21 strain
Dr. Duncan Clark
Duncan at nospam.demon.co.uk
Fri Dec 4 11:58:32 EST 1998
In article <3667E42E.1A3304AA at biocfebs.unizh.ch>, Alexandre Mooser
<amooser at BIOCFEBS.UNIZH.CH> writes
>When I isolate the DNA from XL-I-Blue, everything looks ok (bands at
>right height on DNA gel). Sequencing is ok (right frame). I transformed
>then BL21(DE3) cells with this same DNA (same clone), and isolated the
>DNA to check if everything is still ok.... and problems appeared: the
>constructs seem to be degraded (bands 30% shorter on agarose gel). Of
>course, my protein is not expressed (checked on SDS-PAGE) !!.
>Very frustrating ! I repeated the whole many times, and the same happens
>each time. Can anybody help me ??
>Did anybody already work with BL21(DE3) cells ??
>Any suggestion welcome
This is fairly common for BL21(DE3) constructs. Your protein appears to
be toxic to the cell so it is deleting/rearranging your plasmid. The
problem is leaky expression of T7 RNA polymerase in uninduced cultures
such that you then get a low but sufficiently toxic expression of your
Try BL21(DE3) pLysE or pLysS. The pLysE or S derivatives produce T7
lysozyme which mops up any low level expression of T7 RNA pol and
thereby reduces the toxic expression you are seeing. pLysE produces more
T7 lysozyme than pLysS.
A further alternative is to use Bl21 with the lambda phage CE6. CE6
produces the T7 RNA pol upon infection and hence your protein gets
expressed only in the presence of CE6.
I would try the pLys derivatives first, available I think from Novagen.
Other tricks. Grow your BL21(DE3) recombinant to only low density before
you induce. Never grow them overnight to stationary. Grow at lower
temperature ie 20 or 25C. Change your vector so that it has one or more
lacI repressor sequences between T7 promoter and rbs. This should reduce
the basal expression without IPTG present. Grow in media without yeast
extract as yeast extract has an inducer like substance.
The problem with being on the cutting edge is that you occasionally get
sliced from time to time....
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