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Sepharose 4B两步亲和层析纯化MBL-MASP复合物．再以Sephacryl S-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Western blotting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 degrees Celsius 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.