Development of a method for purifying trypsin that combines precipitation of protein-polyelectrolyte complexes with hydrophobic interaction chromatography.

BRAIA, MAURICIO; TUBIO, GISELA; ANDREWS, BARBARA; SALAZAR, ORIANA; LIENQUEO, MARIA E; ROMANINI, DIANA

New Port

Conferencia; 17th International Conference on Biopartitioning and Purification BPP.; 2013

Development of a method for purifying trypsin that combines precipitation of protein-polyelectrolyte complexes with hydrophobic interaction chromatography. Mauricio Braia [1], Gisela Tubio [1], Oriana Salazar [2], María E.Lienqueo [2] and Diana Romanini [1] (mlienque@ing.uchile.cl) [1] Universidad Nacional de Rosario, Argentina, [2] Universidad de Chile. Trypsin (TRP) is a serine protease widely used in the food and pharmaceutical industries and in molecular biology. Alginate (ALG) is an anionic polylectrolyte obtained from brown algae which have been extensively used for many biomedical applications due to its biocompatibility, low toxicity, relatively low cost and gelation capacity. Proteins and polyelectrolytes form insoluble complexes via electrostatic interactions that might be useful to isolate and immobilize enzymes. Hydrophobic interaction chromatography (HIC) is a powerful technique used for separating proteins based on their hydrophobic properties. The combination of precipitation of protein-polyelectrolyte complexes and HIC is presented as a novel strategy for purifying proteins. The aim of this work was to develop a method for purifying TRP that combines precipitation of TRP-ALG complexes and HIC. At pH 3.50 TRP and ALG interacts to form an insoluble complex that can be precipitated, obtaining a recovery of 93 %. Then, the precipitated complex was dissolved in buffer TRIS 50 mM pH 8.00 NaCl 1 M and injected into a Phenyl-sepharose column. The recovery was 57 % and we are working to improve it. The most important feature is that it allowed separating the TRP from the ALG. Authors would like to thank MINCyT (Argentina) and CONICyT (Chile) for the financial support (CH/11/16).