Construction of an artificial cell membrane anchor using DARC as a fitting for artificial extracellular functionalities of eukaryotic cells

The silent Duffy antigen/receptor for chemokines (DARC) is a receptor-like membrane protein of erythrocytes and mediates no cell transduction not at least regarding a missing or truncated G-loop and therefore it seemed to be the candidate for our cell membrane anchor.We isolated the genetic information of DARC from human genomic DNA and cloned it in a mammalian cell line as a fusion protein via a suitable plasmid vector.In this report we demonstrate that the human plasma membrane protein DARC can be used as an artificial anchor molecule in cell surface engineering applications. We constructed the fusion protein SNAP-tag-DARC, consisting of DARC and the self-labeling protein tag SNAP-tag？ (Covalys). The SNAP-tag？ served as an example for a molecular-technological developed protein that is artificially attached to the extracellular side of the plasma membrane through our DARC-anchor. SnapTag should serve as an example for any extracellular entity and was easy to detect by a commercial detection system. The synthesis of SNAP-tag-DARC, its correct incorporation into the cell membrane and the functionality of the SNAP-tag？ were verified by RT-PCR, Western blotting and confocal fluorescence microscopy and showed the desired functionality as an membrane anchor for an extracellular application entity.Currently desired manipulations of eukaryotic cells comprise the specific modification of their extracellular surface, in particular the cell membrane. Cell membranes can be engineered by attaching new molecules or by modifying or removing their natural components. Of particular interest are proteins, peptides, oligosaccharides and small organic molecules. In combination with other technologies, such modifications of the cell membrane shall enable a range of applications, as cell adhesion in general, single cell arrays, the sorting of cells on surfaces, the manipulation of migration and differentiation in cell cultures, the connection of naturally incompatible cell types, the m