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Team:Bielefeld-Germany/Results/S-Layer/Guide/4a
From 2011.igem.org
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=Small scale cell disruption= | =Small scale cell disruption= | ||
| - | [[Image:IGEM-Bielefeld2011-Sonifier.JPG| | + | [[Image:IGEM-Bielefeld2011-Sonifier.JPG|430px|thumb|left|A Branson Sonifier 450 which we used to disrupt our cells in small scale.]] [[Image:IGEM-Bielefeld2011-Centrifuge.JPG|430px|thumb|left|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 Tris-HCl .... 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 | + | <br style="clear: both" /> |
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| + | 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 Tris-HCl .... 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. | ||
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| + | Only purified S-layer proteins will self-assemble - [[Team:Bielefeld-Germany/Results/S-Layer/Guide/5 | click here for further purification steps]]. | ||
Revision as of 21:01, 27 October 2011
Small scale cell disruption
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 Tris-HCl .... 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.
