Protein Production and Labeling. 11, The soluble extracellular segments of DC-SIGN and DC-SIGNR, which faithfully replicated the tetrameric structure and glycan binding properties of full length lectins, were expressed as inclusion bodies in E. coli and purified by mannose-Sepharose affinity column followed by Superdex size exclusion column as reported previously. The mutant proteins, DC-SIGN Q-274C and DC-SIGNR R278C, were constructed by site-directed mutagenesis and labeled with Atto-643 maleimide as described previously. The labeled proteins were purified by mannose-Sepharose affinity columns. All the proteins were characterized by high-resolution mass spectroscopy (HRMS, see SI, Figs. and). The dye labeling efficiency (per protein monomer) was determined to be ∼82% and ∼90% for DC-SIGN and DC-SIGNR, respectively, based on the relative peak areas of the labeled and unlabeled protein peaks measured by HR-MS (see SI, Fig. ).