E.coli cultures lysing after induction
bpgm at unixg.ubc.ca
Tue Aug 20 11:25:39 EST 1996
I've run into something similar trying to express a fungal protein in E.
coli; IPTG is not required for a decent level of translation.
1. the wild-type leader peptide has an Arg at the -4 position and the Leu
mutant of this lyses the cells when they are cultured at 37; they look
fine when cultured at 30.
2. preliminary results indicate that this also occurs when the fungal
leader is replaced with that from E. coli cytochrome b562.
3. this doesn't occur with the wild-type, or constructs lacking a signal
peptide or having the b562 coding region inserted anti-sense (which still
gives a continuous reading frame for the mature fungal portion).
4. None of the constructs show periplasmic export.
The most obvious explanation to me is that the inner membrane is being
disrupted by translocation problems. Are you trying similar things?
Dr. W. Brent R. Pollock
Dept. of Biochem. & Mol. Biol.
Univ. of B. C.
In article <4vc63d$qbl at netty.york.ac.uk>,
Peter Ashton <pda100 at york.ac.uk> wrote:
>We have a strain of E.coli that we have been using for a few weeks now
>without any problems. Suddenly, the cultures we are growing have started
>lysing whenever they are induced with ITPG. The bulk of the lysis seem to
>occur within the first hour after induction.
More information about the Methods