Patent ID: 7956167
Filing Date: 2011-06-07
Classification: C07K,C12N

Abstract:
1. A method for purifying Clostridium histolyticum collagenase type I and type II proteins from a complex mixture comprising the steps of providing the complex mixture dissolved in an aqueous liquid phase, forming a precipitate of collagenase type I and type II proteins by dissolving ammonium sulfate in the liquid phase, separating the precipitate from the liquid phase, dissolving the separated precipitate in an aqueous buffer comprising Ca2+ ions and having a pH between 6.0 and 8.0, and adjusting the conductivity of the buffer with the dissolved precipitate to a value between 50 and 300 mS/cm, thereby forming a complex buffered solution comprising collagenase type I and type II proteins, reducing clostripain proteolytic activity by a factor of between 90 and 150 and reducing tryptic activity by a factor of between 100 and about 400 relative to the respective proteolytic activities in the complex mixture dissolved in the aqueous liquid phase, extracting the complex buffered solution by contacting it with a hydrophobic stationary phase and adsorbing collagenase type I and type II proteins to the stationary phase, separating the hydrophobic stationary phase with the adsorbed collagenase type I and type II proteins from the extracted solution, and eluting the collagenase type I and type II proteins from the stationary phase, thereby purifying the collagenase type I and type II proteins.