prok. expression

[Michael Cooley]

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..
:  


I don't have any experience with instability at -70, but I have dealt 
with unstable expression in Agrobacterium. E. coli is reasonably stable 
at +4 oC. I kind of doubt the instability is due to temp, but you might 
try storage at other temperatures. What are you adding for a cryogen, 
DMSO or glycerol? For my work I was forces to repeatedly select for high 
expression from single colonies. Eventually I found an improvement.


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