Team:Bielefeld-CeBiTec/Hardware

Overview

Purification Column EluX

One of the non-canonical amino acids (ncAAs) we decided to integrate into our toolkit is 2-nitro-phenylalanine (2-NPA). This ncAA is very interesting to us, as it has the ability to induce a cleavage of the peptide backbone when radiated with light of ʎ = 365 nm wave length. This promises a wide field of possible applications. To demonstrate this astonishing feature, we use this ncAA tointroduce a protein purification system with light-induced elution as an alternative to protease cleavage sides. To put this idea in practice, we started the modelling of a purification column in SketchUp and constructed it with 3D-printed parts and acrylic glass. In this development process, we integrated many advices from professors and experts to render the model as efficiently as possible.
To verify the elution as easily as possible, we created a fusion protein containing GFP for a visible fluorescence signal and streptavidin to immobilize the protein in our column using biotin. The two protein parts are connected through a flexible glycine-glycine-serine-linker of eleven amino acids, in which the sixth amino acid is our 2-NPA. This ncAA will then induce the cleavage of our target protein (GFP) from the streptavidin compound in the purification process.