CAMPERI Silvia Andrea
congresos y reuniones científicas
AN INTEGRATED PROCESS FOR TRYPSIN ISOLATION FROM BOVINE PANCREAS USING BENZAMIDINE OR CTPR IMMOBILISED ON EXPANDED BEADS
N .B. IANNUCCI; G.J. ALBANESI; M. M. MARANI; H.M. FERNÁNDEZ LAHORE; O.CASCONE; S. A. CAMPERI
Village Rio das Pedras, Brasil
Congreso; Enpromer 2005; 2005
Trypsin is a pancreatic serine protease with substrate specificity based on its positively charged lysine and arginine side chains. Its recovery and purification from pancreas is of industrial importance. Partially purified preparations of trypsin are used in the food functionality and in the leather industry. With stringent purity criteria, it is applied in the pharmaceutical industry as a digestive aid. A high-purity product is utilised in the cell culture industry, in proteomics and in the preparation of immunoglobulin fragments. Owing to the proneness of trypsin to undergo autodigestion during purification, the development of rapid and easy-to-scale-up purification methods is of special interest. Conventional isolation and purification of proteins from mammalian tissue homogenates involves a complex sequence of clarification, recovery and purification steps, this resulting in a significant loss of the desired product. Expanded bed adsorption (EBA) with affinity matrices combines clarification, coarse and fine purification in only one step. In this work we report a method for trypsin affinity purification from a bovine pancreas homogenate using the peptide CTPR or p-aminobenzamidine (PAB) immobilised on a particulate matrix suited for expanded bed adsorption techniques (StreamlineTM). Trypsin was purified from a crude bovine pancreas extract by affinity chromatography in the expanded-bed adsorption mode. By using a clarified extract, maximum capacity for CTPR-Streamline was 47.4 mg/ml and for PAB-Streamline 78.9 mg/ml while Kd values were 0.39 and 0.38 respectively. Dynamic capacity was 23.0 and 46.0 mg/ml for CTPR- and PAB-Streamline respectively. When the purification process was applied to unclarified pancreas extract, 80 % trypsin recovery with a purification factor of 18.7 was achieved. Cationic and anionic trypsin obtained from the affinity column were separated by ion-exchange chromatography. The method combines clarification, capture and purification in a single step thus reducing protein purification costs.