Preparative purification process for human furin

Details Preparative purification process for human furin Preparative purification process for human furin

C - Chemistry, Metallurgy

12

N

C12N 9/64 (2006.01) A61K 38/48 (2006.01) C07K 1/18 (2006.01) C12N 15/57 (2006.01)

Patent

CA 2684885

Recombinant truncated human furin was expressed in CHO cells and concentrated approximately 50 -fold by ultrafiltration and diafiltration. The concentrate was purified by column chromatography on Capto-MMC~ resulting in a 30-50 fold purification factor and a yield of at least 60%. The at least 20% pure preparation obtained after Capto-MMC~ chromatography had already a purification degree allowing on-column maturation of pro-VWF. Then an additional Arginine Sepharose chromatography purification was carried out. This two column process for purification of truncated human furin resulted in an almost pure furin preparation with a specific activity of approximately 290,000 U furin/mg protein and a yield of about 50%.

Selon l'invention, une furine humaine tronquée recombinée est exprimée dans des cellules CHO et concentrée environ 50 fois par ultrafiltration et diafiltration. Le concentré est purifié par chromatographie sur colonne Capto-MMC~, résultant en un facteur de purification de 30 à 50 fois et un rendement d'au moins 60%. Les 20% au moins de préparation pure obtenue après la chromatographie Capto-MMC~ présentaient déjà un degré de purification permettant une maturation sur colonne de pro-VWF. Ensuite, une purification chromatographique supplémentaire d'arginine sépharose est réalisée. Ces deux processus colonnaires de purification de furine humaine tronquée permettent d'obtenir une préparation de furine presque pure présentant une activité spécifique d'environ 290 000 U furine/mg de protéine et un rendement d'environ 50%.

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