Scott Bruce Mulrooney
mulrsb at umich.edu
Sat Feb 18 13:39:57 EST 1995
mahesh (mahesh.sitaram.1 at nd.edu) wrote:
: HI, I have been trying to express a protein called lactoferrin(Lf) in
: prokaryotes. Lf has not been expressed in prokaryotes in literature.
: Tried a variety of expression vectors and cell strains. The only
: combination that worked was the expression vector pGEMEX-2 in
: BL21(DE3)-gangbuster expression with protein in the soluble fraction!!
: seemed like the Gods had finally smiled... So made -70 stocks of the
: expressing cells. The fun started six mos. later when I tried using the
: -70 sotcks to express protein..
: -frozen stocks no longer express
: -only BL21(DE3) freshly transformed with the Lf cDNA express. However
: the expression is about 100 times reduced and sporadic, i.e. sometimes they
: express and sometimes they do not.
: --70 stocks of the newly expressing cells (with considerably reduced
: expression) do not express even if they're only a week old.
: -Q: Have you come across anything like this. If so any suggestions..
Someone that I work with had similar problems using the
T7 system: it was finally traced to plasmid instability.
Liquid cultures started from a single colony on a plate
would not express the gene; but when a large scraping from
a plate was used to start the liquid cultures, there was
expression. My suggestion is to start with a freshly transformed
cology, restreak on a plate, and start a liquid culture using
a large scraping from this plate.
Are you using ampicillin as the antibiotic? If so, it is known to
be bad for maintaining some plasmids. The problem is that the beta-
lactamase is in the periplasm of the E coli, which causes very
rapid depletion of the amp from the growth medium. By the time the
culture is slightly turbid, most of the ampicillin is gone (the
review of the T7 system in v. 185 of Meth. Enzymol. points this out).
There are two easy ways to check for plasmid instability; 1) do cells
grown that do not express the protein have the plasmid? - save a ml
of the cells and do a plasmid prep, and 2) test the cells at the time
of harvest - make appropriate dilutions of the culture and plate on
plain LB and some on LB plus amp. If there is plasmid loss during
growth, the LB plus amp titer of cells will be lower.
University of Michigan
Department of Biological Chemistry
mulrsb at umich.edu
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