沙雷氏蛋白酶MP及其抑制剂LupI的复合物纯化及结晶

Purification and Crystallization of Complex Serralysin-Like MP and Its Inhibitor LupI

沙雷氏蛋白酶属于锌金属蛋白酶M10B亚家族,是一些细菌性疾病的关键致病因子。专一性沙雷氏蛋白酶抑制剂在体外可靶向抑制沙雷氏蛋白酶,从而减弱产沙雷氏蛋白酶的细菌病原体的活性,成为疾病治疗的新思路。沙雷氏蛋白酶MP和其抑制剂Lup I均来源于海洋微生物Flavobacterium sp. YS-80-122,本研究分别纯化了蛋白酶MP、抑制剂LupI及MP-LupI的复合物,对MP-LupI复合物进行结晶条件筛选,成功在2种条件下获得MP-LupI复合物晶体,并经X-射线衍射收集到分辨率达2A的高质量衍射数据,其晶胞空间为P1211,晶胞参数为a=51.66A、b=51.85A、c=102.14A、α=γ=90°、β=97.68°。

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 95μl 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 2A.The unit cell space was P1211.The unit cell parameters are a=51.66A,b=51.85A,c=102.14Å,α=γ=90°andβ=97.68°.