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Team:Bielefeld-Germany/Results/S-Layer/Guide/4b
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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 leads to monomeric S-layer proteins. The cell disruption is carried out in a buffer containing 6 M urea, 50 mM | + | 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 leads 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. |
Commonly used lab methods like sonification or enzymes for cell disruption are unpracticable when you have to disrupt bigger amounts of biomass. In this case, mechanical methods like pebble mills or high-pressure homogenizers are the procedures of choice. But mechanical application of energy always leads to a heat input. Since heat can damage your proteins, you have to ensure a sufficient cooling of the solution during cell disruption. We achieved this by placing our high-pressure homogenizer in the cooling chamber of our lab and not running it continuously, but in cycles (3 cycles with cooling phases between the cycles, p = 800 bar). | Commonly used lab methods like sonification or enzymes for cell disruption are unpracticable when you have to disrupt bigger amounts of biomass. In this case, mechanical methods like pebble mills or high-pressure homogenizers are the procedures of choice. But mechanical application of energy always leads to a heat input. Since heat can damage your proteins, you have to ensure a sufficient cooling of the solution during cell disruption. We achieved this by placing our high-pressure homogenizer in the cooling chamber of our lab and not running it continuously, but in cycles (3 cycles with cooling phases between the cycles, p = 800 bar). | ||
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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]]. | Only purified S-layer proteins will self-assemble - [[Team:Bielefeld-Germany/Results/S-Layer/Guide/5 | click here for further purification steps]]. | ||
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Latest revision as of 02:46, 29 October 2011
Cell disruption with a high-pressure homogenizer
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 leads 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.
Commonly used lab methods like sonification or enzymes for cell disruption are unpracticable when you have to disrupt bigger amounts of biomass. In this case, mechanical methods like pebble mills or high-pressure homogenizers are the procedures of choice. But mechanical application of energy always leads to a heat input. Since heat can damage your proteins, you have to ensure a sufficient cooling of the solution during cell disruption. We achieved this by placing our high-pressure homogenizer in the cooling chamber of our lab and not running it continuously, but in cycles (3 cycles with cooling phases between the cycles, p = 800 bar).
The cell debris is removed with a centrifugation step where the supernatant is decanted and used for further purification.
Only purified S-layer proteins will self-assemble - click here for further purification steps.
