Reconstitution of (1→3)-β-D-glucans measurement system using recombinant Limulus polyphemus Factor G

Horseshoe crab Factor G is a heterodimeric serine protease zymogen that is activated by (1→3)-β-D-glucans (BDG) from fungal cell walls. This reaction is used in diagnostic agents for deep-seated mycosis. At present, functional analysis using Factor G from Tachypleus tridentatus has been performed, and genetic information has been published, but reconstitution using recombinant proteins has not yet been achieved. In this study, we cloned the genes for Factor G α and β from Limulus polyphemus; two gene sequences were obtained for Factor G α and seven for β. The obtained L. polyphemus Factor G α was used to specifically remove BDG from the culture medium for eliminating the activator BDG. The optimal combination for each sequence was examined with BDG removal medium, and a combination was found that featured BDG-dependent activity. These results indicate that a BDG assay system using recombinant Factor G is feasible in reconstitution. This research will support future reagent development that does not require natural horseshoe crab resources. KEY POINTS: • Cloned novel Factor G α subunit and β subunit genes from L. polyphemus • Proposed a method of removing BDG without reducing culture medium performance • Identified combination of recombinant α and β subunits for BDG-dependent activation.