Abstract:Proteases are a class of enzymes that hydrolyze proteins by cutting protein peptide bonds in vivo, controlling protein size, composition, spatial conformation, and their final degradation. Physiological activities and diseases in organisms are closely related to proteases, such as those for digestion and absorption of food, blood coagulation, hemolysis, anti-inflammatory, blood pressure regulation, cell differentiation, cancer metastasis, and activation of physiologically active peptides. Serralysin-like belongs to the subfamily of zinc metalloprotease M10B and secretes by a variety of pathogenic gram-negative bacteria. It is a key virulence factor for some diseases. Serralysin-like inhibitor can the inhibit target serralysin-like in vitro. The inhibition of bacterial virulence factors presents an antimicrobial strategy that is non-destructive, by attenuating virulence mechanisms without directly challenging bacterial cell viability. The serralysin-like MP and its inhibitor LupI studied in this paper are all secreted by the marine microbial Flavobacterium sp. YS-80-122. We purified the protease MP, inhibitor LupI and MP-LupI complexes. The protease MP was purified by affinity chromatography while the inhibitor LupI was purified by size exclusion chromatography and ion exchange chromatography. After being tested and concentrated, the two were mixed according to the equimolar ratio to obtain the MP-LupI complexes. Finally, purified MP-LupI complexes by size exclusion chromatography to obtain an electrophoresis-purified, single-purification sample of about 95 μl of MP-LupI complexes with a protein content of 20 mg/ml. The MP-LupI complexes were crystallized and successfully obtained under two conditions: 0.1 mol/L DL-malic acid, pH 7.0, 12% (w/v) PEG 3350 to obtain a rhombohedral crystal of MP-LupI complex and 0.2 mol/L NaCl, 0.1 mol/L MES, pH 6.5, 10% (w/v) PEG 4000 to obtain an prism crystal of the MP-LupI complex. The X-ray diffraction data was collected from the Shanghai source to obtain high-quality diffraction data with a resolution of 2 Å. The unit cell space was P1 21 1. The unit cell parameters are a = 51.66 Å, b = 51.85 Å, c = 102.14 Å, α=γ=90° and β=97.68°.
