Abstract
Dectin-1 (CLEC7A), a C-type lectin-like receptor that recognizes β-1,3 glucans, has a key role in the innate immune system. While the lectin domain of mouse Dectin-1 has been solubilized and refolded from inclusion bodies in Escherichia coli, similar refolding of the human Dectin-1 lectin domain is hindered by the formation of misfolded multimers with aberrant intermolecular disulfide bonds. The aim of this study was to develop a method for the large-scale production of the human Dectin-1 lectin domain. Based on a protocol for the murine domain, the human Dectin-1 lectin domain was expressed as a fusion protein with Protein G B1, a solubility-enhancing tag. The refolding and purification conditions were then optimized by testing a range of buffers with and without Ca2+ ions. The inclusion of 1 mM Ca2+ ions in both the refolding and purification buffers resulted in high yields of a monomeric form of the human Dectin-1 lectin. The resulting recombinant protein was demonstrated to be functional, showing specific binding to the β-glucan laminarin as verified by thermal shift assays, gel filtration chromatography and NMR. Furthermore, NMR experiments revealed that the human Dectin-1 lectin domain binds Ca2+ ions. The recombinant protein will support structural biology studies to clarify differences in β-glucan binding specificity between human and mouse Dectin-1, and to explore the effects of mutations on the functionality of human Dectin-1.
Published Version
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