人血浆中MBL-MASP复合物的纯化与分离

目的 从人血浆中分离纯化甘露聚糖结合凝集素(MBL)和MBL相关丝氨酸蛋白酶(MASP)。方法 用非活化Sepharose4B两步亲和层析纯化MBL-MASP复合物,再以SephacrylS-300柱凝胶过滤将MBL和MASP分离。在纯化MBL-MASP复合物的整个过程中,除凝胶过滤缓冲液外,均加入丝氨酸蛋白酶抑制剂苯甲磺酰氟(PMSF)和金属蛋白酶抑制剂1,10-邻二氮杂菲(1,10-phenanthroline),并控制温度在4 ℃。结果 获得高度纯化的MBL和酶原形式的MASP。SDS-PAGE和Westernblotting表明所纯化的MBL相对分子质量为28000和32000肽链构成的功能性多聚体;配体结合测定和酵母菌凝集试验证实所纯化的MBL具有生物学活性。结论 建立了一种比较简便的同时分离纯化MBL和MASP酶原的方法。

Purification and separation of mannan-binding lectin (MBL) and MBL-associated serine proteases complex from human plasma

Objective To separate mannan-binding lectin (MBL) and MBL-associated serine proteases (MASPs) from human plasma. Methods A two-step affinity chromatography on underivatized sepharose 4B was employed for purification of MBL-MASP complex, followed by gel filtration on a Sephacryl S-300 column for separation of MBL and MASPs from the complex. The purification procedures were performed at 4℃ with the addition of two proteolytic inhibitors, phenyl methylsulfonyl fluoride and 1,10-phenanthroline during affinity chromatography but not in the gel filtration buffer. Results Preparations of highly purified MBL and proenzyme MASPs were obtained. The purified MBL was shown by SDS-PAGE and Western blotting to be a functional multimer composed of 28 000 and 32 000 peptide chains, with high bioactivity as demonstrated by ligand-binding assay and yeast agglutination experiment. Conclusion A simple and convenient procedure is established successfully for the purification of MBL and the proenzyme MASPs.