Studies of protein partition in non conventional aqueous two-phase systems as method to purify trypsinogen and alpha-chymotrypsinogen from bovine pancreas

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• 作者：Luciana Pellegrini Malpiedi^a ; Guillermo A. Picó ; ^a ; Bibiana B. Nerli ; ^a ; ^{bnerli@fbioyf.unr.edu.ar}
• 关键词：Trypsinogen ; Alpha-chymotrypsinogen ; Aqueous two-phase systems ; Proteins partitioning
• 刊名：Separation and Purification Technology
• 出版年：2011
• 出版时间：24 March 2011
• 年：2011
• 卷：78
• 期：1
• 页码：91-96
• 全文大小：710 K

文摘

Aqueous two phase systems (ATPSs) formed by polyethyleneglycol (PEG) and sodium tartrate (NaTart) pH 5.00 are non conventional systems of low impact. Their ability to purify trypsinogen (TRPz) and alpha-chymotrypsinogen (ChTRPz) from bovine pancreas was evaluated. The effect of several variables such as molecular weight of PEG, tie line length and phase–volume ratio on zymogen partitioning was explored. Those systems formed by PEG of molecular weight 600 (20.62 % , w/w) and NaTart (12.85 % , w/w) with a phase–volume ratio equal to one were found to evidence the best purification performance, and were employed to design a two-stage purification process.

Firstly, a ATPS extraction step yields both to recover most of pancreatic TRPz and ChTRPz (76 % and 87 % respectively) in the PEG600 enriched-top phase and to eliminate other pancreatic homogenate components – cell debris, nucleic acids and lipids – which either partition into the bottom phase or precipitate at the interface.

Then, the top phase – from this first extraction step – is mixed with a given quantity of PEG6000 in order to induce the formation of a new aqueous two phase system in which the back extraction of both zymogens takes place. This second stage allows to obtain a mixture of both zymogens highly pure (95 % ) in a bottom phase with low PEG concentration (3.70 % , w/w) and therefore suitable for being used in multiple applications.