Why are my E. coli cells not pellet-able? - when expressing a chimeric recombinant protein (Feb/25/2009 )
Hey,
I'm expressing a lipopolysaccharide biosynthetic enzyme in E. coli. This is a enzyme from C. burnetii chimerized with a short C-terminal sequence from its E. coli homolog. The recombinant protein expressed as cytosolic oluble protein in E. coli BL21(DE3). But interestingly, comparing to the wild-type C. burbetii enzyme, this protein seems to make the E. coli cells unable to pellet well.
Wild-type C. burnetii enzyme/BL21(DE3):
-- Spin down at 6500 g for 10 min after fermentation overnight.
-- Formed nice pellet in the centrifuge bottle.
C. burnetii enzyme hybridized with a E. coli tail/BL21(DE3):
-- Spin down at 6500 g for 10 min after fermentation overnight.
-- Cell looks unsticky to each other and can't pellet down.
This happened several times. What would make the E. coli cells non-sticky? A change in morphology? Changes on the outer membrane? Anyone has any thought?
Thanks,
Lydia
change in cell density. Change in the number of cells in solution, are also possible alternatives.
Thanks. Lower density or higher density would make it non-sticky?
I grew the cells at 37'C overnight and obtained a reasonably high amount of cells from 500 mL LB/amp, compared to normal fermentation of cells expressing the wild-type protein.
perneseblue on Feb 25 2009, 11:38 AM said:
The cell are being pelleted by a centrifuge. Stickiness / clumping... while a factor isn't the only one. The density of the cells and the number (thus the pellet size, after x time of centrifuging) also play a role.
You could try increasing the centrifuge speed. Or spin for longer.
We had a mutant that over-produced an exopolysaccharide. We could not, for the life of us, get this strain to pellet; this phenotype was actually how we found what proved to be a very interesting EPS locus and regulatory mechanism in our bug.
Hey, HomeBrew,
That's very interesting! Can you give me the citation of your paper published on that?
Thank you for your input!
Lydia
HomeBrew on Feb 25 2009, 05:01 PM said:
See here.