Team:Freiburg/Description

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This is the wiki page
of the Freiburger student
team competing for iGEM 2011.
Thank you for your interest!

Contents

Precipitator

Precipitator binding polystyrene surface with the plastic binding domain and a His-tagged Protein via Nickel

We created a cellular, self-replicating purification device for His-tagged proteins. It is a completely artificial fusion protein, which consists of a repeating LRRNT motif domain, coordinating Ni2+ Ions on its surface capped on N and C terminal ends by a hagfish sequence of a similar LRRNT motif. A second domain, a short hydrophobic peptide stretch, binds a polystyrene surface. We named this construct “THE PRECIPITATOR”. After expression of the Precipitator in a light inducible E. coli strain, the cells are lysed and the lysate is taken up by a serological pipette, in preparation of the actual protein purification steps. The underlying mechanism is comparable to Ni-NTA columns. Our Precipitator protein binds on the surface of the pipette, presenting the chelated Nickel ions. Free coordination sites of the Nickel ions are exposed, so that a His-tagged protein can attach to them. Cells expressing a His-tagged protein can be dissolved by the heat inducible lysis device. Subsequently, when the lysate is taken up with a serological pipette coated with the Precipitator protein, the His-tagged proteins bind to it. Cell debris is then washed off, while the His-tagged protein stays and is eluted afterwards, in the same fashion as done in Ni-NTA columns with imidazole solutions, increasing in concentration. The His-tagged protein is finally captured in a distinct fraction.

Green light receptor

Blue light receptor

Red light receptor

Lysis cassette

                       

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