Detailed AFM Force Spectroscopy of the Interaction Between CD44–IgG Fusion Protein and Hyaluronan
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Atomic force microscopy (AFM) force spectroscopy was used to study the single-molecule rupture events of the interaction between hyaluronan (HA) and the binding domain of its cell surface receptor CD44. AFM probes were amino terminated with 3-aminopropyl triethoxy silane (APTES) followed by covalent coupling of protein A, enabling the binding of the CD44–HA-binding domain, as part of a CD44–Fc fusion protein. HA was covalently bound to APTES-coated silicon surfaces. Single-rupture events were recorded at various loading rates revealing an energy barrier: E b = 24 ± 1 kT and characteristic distance: x β = 1.3 ± 0.1 nm for this interaction. This quantification will be of interest in applications and research involving the use of the CD44–Fc fusion protein since we observe a weaker interaction between HA and CD44–Fc than what has been reported for the entire native CD44 molecule.
KeywordsCD44 link domain Fusion protein Hyaluronan Force microscopy
We acknowledge Bram van den Broek (Leiden University, The Netherlands, currently at the Netherlands Cancer Institute, The Netherlands) for the fruitful discussions and Jesper Koning (Delft University of Technology and Leiden University, The Netherlands) for his experimental support. This work was financially supported by a VENI grant from the Netherlands Organization for Scientific Research (NWO, grant no. 700.56.412) to LCPMdS.
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