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Purification of Trypsinogen from Bovine Pancreas by Combining Aqueous Two-Phase Partitioning and Precipitation with Charged Flexible Chain Polymers
PELLEGRINI, LUCIANA; ROMANINI, DIANA; NERLI, BIBIANA; PICÓ, GUILLERMO
Conferencia; International Conference on Biopartitioning and Purification; 2007
Universidad de Minho-Portugal
Purification of Trypsinogen from Bovine Pancreas by Combining Aqueous Two-Phase Partitioning and Precipitation with Charged Flexible Chain Polymers Luciana Pellegrini Malpiedi, Diana Romanini, Bibiana Nerli, Guillermo Picó Bioseparation Lab. Physical Chemistry Department. Faculty of Biochemical and Pharmaceutical Sciences. University of Rosario, FonCyT and CONICET. Suipacha 570 (S2002RLK) Rosario. ARGENTINA. E-mail: firstname.lastname@example.org Increasing quantities of highly pure trypsin are required to fulfil the needs of both biopharmaceutical manufacturers and food industries. The goal of this work is to optimise the conditions to purify trypsinogen (TRPz), the trypsin inactive precursor, from bovine pancreas by applying a macro-scale method: the liquid-liquid extraction with aqueous two-phase systems (ATPSs). In a previous study, we demonstrated that systems formed by polyethyleneglycol of molecular weight 1,450 (PEG1450) and sodium citrate, pH 8.2 (NaCit) showed a high capability for isolating TRPz from the pancreatic homogenate . TRPz partitioned to the citrate-rich phase while the other proteins partitioned towards the PEG-rich phase. In this work, another variables such as the tie line length (TLL), the biomass concentration and the phase-volume ratio (Vr = VTop / VBottom) were analysed. Besides, a precipitation step with a charged polymer was also assayed in order to improve the purification process. We selected polyethyleneimine (PEI), a positively charged flexible chain polymer, which has the ability to precipitate negative molecules such as nucleic acids and acid proteins . Different final PEI concentrations were assayed (0.10; 0.25 and 0.50 % w/w). The effectiveness of selective precipitation was evaluated by measuring both the ratio between the absorbances at 260 and 280 nm and the TRPz content. By using a final PEI concentration of 0.25 % w/w , the 55 % of nucleic acids were removed without a significant loss of TRPz content. The resulting supernatant was clear, practically free of insoluble particulate material from the tissue disruption and presented such a low viscosity that the subsequent processing was facilitated. A higher PEI concentration was inconvenient since it caused a significant precipitation of both the nucleic acids and the TRPz. Increasing supernatant masses (up to 5, 7.5 and 10 % of the total system mass respectively) were partitioned in ATPSs with different compositions and volume-ratios. Systems corresponding to a long TLL showed higher TRPz recoveries (R) and purification factors (PF) than those of a short TLL. Both an enhancement of the TRPz recovery from 50 to 90 % and a decrease in the PF from 3.7 to 2.5 were observed when the partitioned sample was increased. On the other hand, by reducing the Vr to 0.5, the PF was enhanced to 4.6, the R being 84 %. Our results show that the selective precipitation with PEI followed by partitioning in PEG/Cit ATPSs may be considered as a suitable strategy for purifying TRPz from pancreatic homogenate. This has several advantages which can be summarized as follows: a) ease of scale-up; b) low material costs; c) the possibility of polymer recycling and d) the biodegradability of citrate anion. ________________  G. Tubio; L. Pellegrini; D. Romanini; B. Nerli; G. Picó. An Improving Method for Purifying a Protease of High Commercial Value, the Bovine Pancreatic Trypsin. Congress Food Science and Food Biotechnology in Devleoping Countries. Saltillo. México. 2006.  BW De Walt; JC Murphy; GE Fox; RC Willson. Compaction agent clarification of microbial lysates. Compaction agent clarification of microbial lysates. Protein Expr. Purif. 28 (2003) 220-223.