proper plasmid vectors for membrane protein fragment

Dominic-Luc Webb molmed domweb at mbox.ki.se
Thu Nov 15 15:08:56 EST 2001


On Thu, 15 Nov 2001, Sean Xie wrote:

   > Hi, I would appreciate it if anyone can give me some suggestions about
   > which commercial or noncommercial plasmid vector systems are suitable
   > for expression membrane protein fragment (with reasonable yield)?


I am learning every day as I go. I have had a nightmare of a
time getting decent yields with the IPTG inducible T7 promoter
system in E coli BL21(DE3) lysogens. However, it would be
ignorant to not point out that many groups are using this
system with success. The pET and pCMX plasmids are what is
used typically. There is a whole science in itself in protein
expression using this system and yields can be outright
pitiful. If you go with a bacterial system, there are some
different views about whether or not there should be a
ribosome site, like a Shine Dalgarno sequence, but my
experience is that you get more protein if you have it.

Novagen supplies kits, but at least a couple of the bugs
I got that were directly from their glycerol stocks did
not have some of the characteristics I was expecting for
DE3 lysogens. I fear that while this is a popular expression
system, you could end up spending a lot of time trying to
get it to work well.

A typical protocol would be something like...

grow to OD600 = 0.5-0.6
centrifuge, add new medium +  antibiotic
add 0.1 to 1 mM IPTG
grow for 3 hours, 37 C.
Harvest.

That is what is shown in the Novagen catalog. I have basically
same system as one of their catalog pictures of a plasmid
bering EGFP. I need a fluorescence spectrophotometer to
detect any signal (i.e., very weak).

The lac promoter is another possibility. I think it was
In vitrogen that cells a tag-less bacterial expression
plasmid that uses lac instead of T7. Not inducible, but
a friend told me she gets "huge" expression of EGFP in
such systems without doing anything to promote transcription.
Of course, T7 is better if your target protein is toxic.
I could mention there are other lysogens, like HMS174(DE3)
one could try if BL21(DE3) does not work (this is what I
am presently doing).

If you go with T7, I should also mention that IPTG
treatment in a cell with functional plasmid should
cause pretty much complete loss of growth, detectable
by optical density. Nice little test for T7 RNApol
induction.

Some groups have gone to yeast systems. I can't
offer any ideas about this, other than I am
thinking to switch to this myself.


Cheers,


Dominic-Luc Webb
Karolinska Hospital/Institute




More information about the Microbio mailing list