title: Single-Step Purification of Monomeric l-Selectin via Aptamer Affinity Chromatography.
abstract: l-selectin is a transmembrane receptor expressed on the surface of white blood cells and responsible for the tethering of leukocytes to vascular endothelial cells. This initial intercellular contact is the first step of the complex leukocyte adhesion cascade that ultimately permits extravasation of leukocytes into the surrounding tissue in case of inflammation. Here we show the binding of a soluble histidine tagged l-selectin to a recently described shortened variant of an l-selectin specific DNA aptamer with surface plasmon resonance. The high specificity of this aptamer in combination with its high binding affinity of ~12 nM, allows for a single-step protein purification from cell culture supernatants. In comparison to the well-established Ni-NTA based technology, aptamer affinity chromatography (AAC) was easier to establish, resulted in a 3.6-fold higher protein yield, and increased protein purity. Moreover, due to target specificity, the DNA aptamer facilitated binding studies directly from cell culture supernatant, a helpful characteristic to quickly monitor successful expression of biological active l-selectin.
authors.affiliation: Institute of Laboratory Medicine, Clinical Chemistry and Pathobiochemistry, Charité-Universitätsmedizin Berlin, 13353 Berlin, Germany. christian.kuehne@charite.de.
authors.forename: Christian
authors.initials: C
authors.lastname: Kuehne
authors.affiliation: Institute of Chemistry and Biochemistry, Freie Universität Berlin, 14195 Berlin, Germany. stefanie.wedepohl@fu-berlin.de.
authors.forename: Stefanie
authors.initials: S
authors.lastname: Wedepohl
authors.affiliation: Institute of Laboratory Medicine, Clinical Chemistry and Pathobiochemistry, Charité-Universitätsmedizin Berlin, 13353 Berlin, Germany. jens.dernedde@charite.de.
authors.forename: Jens
authors.initials: J
authors.lastname: Dernedde
mesh_terms: D002448:Cell Adhesion; D002846:Chromatography, Affinity; D019041:L-Selectin; D007962:Leukocytes; D009841:Oligonucleotides
chemical_list: D009841:Oligonucleotides; D019041:L-Selectin
keywords: ">l-selectin; DNA; SPR; affinity; aptamer; purification; recombinant protein