A recombinant serine protease inhibitor, CrSPI-1, from horseshoe crab, which inhibits subtilisin with a K
i of 10−9
M, has been cloned, expressed, purified and cocrystallized with subtilisin. The crystals diffracted to 2.6 Å resolution.
Serine proteases play a major role in host–pathogen interactions. The innate immune system is known to respond to invading pathogens in a nonspecific manner. The serine protease cascade is an essential component of the innate immune system of the horseshoe crab. The serine protease inhibitor CrSPI isoform 1 (CrSPI-1), a unique nonclassical Kazal-type inhibitor of molecular weight 9.3 kDa, was identified from the hepatopancreas of the horseshoe crab Carcinoscorpius rotundicauda. It potently inhibits subtilisin and constitutes a powerful innate immune defence against invading microbes. Here, the cloning, expression, purification and cocrystallization of CrSPI-1 with subtilisin are reported. The crystals diffracted to 2.6 Å resolution and belonged to space group P21, with unit-cell parameters a = 73.8, b = 65.0, c = 111.9 Å, β = 95.4°. The Matthews coefficient (V
M = 2.64 Å3 Da−1, corresponding to 53% solvent content) and analysis of the preliminary structure solution indicated the presence of one heterotrimer (1:2 ratio of CrSPI-1:subtilisin) and one free subtilisin molecule in the asymmetric unit.