Abstract
The need to functionalize cell membranes in a directed way for specific applications as single cell arrays or to force close cell-to-cell contact for artificial intercellular interaction and/or induction concerning stem cell manipulation or in general to have a tool for membrane and cell surface-associated processes, we envisaged a neutral inactive membrane anchor for extracellular entities to facillitate the above mentioned functionalities.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.
Highlights
Desired manipulations of eukaryotic cells comprise the specific modification of their extracellular surface, in particular the cell membrane
We examined whether the human plasma membrane protein Duffy antigen/receptor for chemokines (DARC) [4] can act as such a nanobiotechnological polypeptide anchor fused with a functional peptide of interest
The chosen DARC was amplified from human genomic DNA by PCR and the synthesis products were ligated in an expression plasmid
Summary
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. The presentation of new molecules on the cell surface can be achieved by attaching them as a fusion partner protein to a transmembrane protein through conventional gene transfer. DARC was chosen because it is functionally redundant and not known to initiate intracellular signal transduction [5]. It is a glycoprotein with the predicted topology of a G protein-coupled receptor. DARC is a silent chemokine receptor that binds several chemokines, but shows no G protein-coupled signal transduction. DARC lacks the typical DRY-motif on the second intracellular loop, which is an essential requirement for signal transduction of all residual polypeptides of the GPCR family [8]
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