Hi Gloria, Yes, chloramphenicol-resistant cells are presumably a problem for this method, so other ribosome-targeting antibiotics (such as tetracycline, as you mention) may work in those cases. We've not tried this, but it would be interesting to see if it works. It also would serve to test the mechanism a bit, as similar behavior would indicate that halting protein translation is the critical aspect. I will add that, in my experience, this method works best when there is some small amount of soluble material detectable on SDS-PAGE. For totally insoluble material, we've not much success, but we also have not explored all of the options discussed in the FEBS Letters reference. 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 [email protected] On 10/17/14 10:05 AM, "Gloria Borgstahl" <[email protected]> wrote: >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 ><[email protected]> 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 >[email protected] > > > > > >On 10/17/14 7:51 AM, "xaravich ivan" <[email protected]> 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 >> >> > > > > > > >
