Thanks Mark, this is a good tip what would you use for Rosetta cells though? Tetracyclin?
On Fri, Oct 17, 2014 at 8:31 AM, Mark Wilson <mwilso...@unl.edu> wrote: > Hi Ivan, > We've had good luck with the addition of chloramphenicol ~1 hour prior to > harvest, as described in: > Carrió, M. M., and Villaverde, A. (2001) Protein aggregation as bacterial > inclusion bodies is reversible. FEBS Lett. 489, 2933. > We usually combine this with lower temperature growth (20 C). > Best regards, > Mark > > Mark A. Wilson > Associate Professor > Department of Biochemistry/Redox Biology Center > University of Nebraska > N118 Beadle Center > 1901 Vine Street > Lincoln, NE 68588 > (402) 472-3626 > mwilso...@unl.edu > > > > > > On 10/17/14 7:51 AM, "xaravich ivan" <xaravich.i...@gmail.com> wrote: > > >Dear cc4bb enthusiasts, > > > >This is slightly off topic but many protein crystallographers might be > >familiar with this problem. > > > > > >I have been trying to over-express a bacterial (non-E.Coli) protein in > >E.Coli and more than 80% goest to inclusion bodies. > > > > > > > >I tried the following > > > > > >Lowering the IPTG concentration to 0.1mM (range test from 0.075 mM - 0.5 > >mM) > > > > > >Cold shock for 30 minutes in ice before induction > > > > > >Slow rotation speed at 18 degrees O/N after induction > > > > > >While these steps helped a bit I still get about 50-60% of my protein in > >inclusion bodies. > > > > > >I would like to know what other steps do you suggest to enhance the yield > >in the soluble fraction (without changing the host strain or manipulating > >the DNA) > > > > > >Thanks in advance > > > >Ivan > > > > >
