From 2011.igem.org

Revision as of 02:34, 29 October 2011

Small scale cell disruption

A Branson Sonifier 450 which we used to disrupt our cells in small scale.

A Sigma 6K15 centrifuge which we used in our project.

The S-layer fusion proteins form inclusion bodies in the E. coli cells (at least most of them). Inclusion bodies have the advantage that they are relatively easy to clean-up and are resistant to proteases. But inclusion bodies are unsoluble so they have to be solubilized by urea or guanidin hydrochloride. In addition, these chemicals suppress the self-assembly ability of the S-layer proteins which leeds to monomeric S-layer proteins. The cell disruption is carried out in a buffer containing 6 M urea, 50 mM phosphate buffer (pH 7.4), 300 mM sodium chloride and 10 mM imidazol. The cells are lyzed by sonification (5 times 2 minutes on ice, max. 20 W, cooling on ice between these steps). Afterwards the lysate is incubated for 1 h at 4 °C on a shaker or vertical rotator to solubilize the inclusion bodies. The cell debris is removed by centrifugation and the supernatant is used for further purification.

Only purified S-layer proteins will self-assemble - click here for further purification steps.