MertensR at JRF.be (MertensR at JRF.be) wrote:
: Does anyone know if there are better expression hosts for the bacterial
: (Escherichia coli) expression vector pMal-p (from New England Biolabs)
: than the TB1 strain?
: Did anyone try a protease-deficient strain?
: I'm expressing G protein coupled receptors and the yield is pretty low
: (40 fmol/1000000000bacteria). If someone knows how to boost the
: production please let me know. (I have tried to change the OD600 for
: induction and the growth time after induction and the temperature...)
I used pMalc2 in TB1, BW401?, SA2817, and two SG### strains (whose numbers
I have forgotten). Susan Gottesman suggested these strains to us; she
has a chapter in Methods of Enzymology on the protease deficients. We
saw far better expression/yield in each than TB1; lowering temperatures
from 37C to 30C also helped expression/yeild. SA2817 (ompT-, and one
other genotype that slips my mind) worked best; we hypothesized that
outer membrane proteins were shredding our protein (MBP-MGA from
Strep. Pyogenes) when the cells burst. Also, protease inhibitors are an
option; but, they run the risk of inhibiting your protein's activity.
Andrew S Heath ash at umich.edu
