Features of the acid protease partition in aqueous two-phase systems of polyethylene glycol-phosphate: chymosin and pepsin

The partitioning of chymosin (from Aspergilus niger) and pepsin (from bovine stomach) was carried out in aqueous-two phase systems formed by polyethyleneglycol-potassium phosphate. The effects of polymer concentration, molecular mass and temperature were analysed. The partition was assayed at pH 7.0 in systems of polyethyleneglycol of molecular mass: 1450, 3350, 6000 and 8000. Both proteins showed high affinity for the polyethyleneglycol rich phase. The increase of polyethyleneglycol concentration favoured the protein transfer to the top phase, suggesting an important protein-polymer interaction. Polyethyleneglycol proved to have a stabilizing effect on the chymosin and pepsin, increasing its protein secondary structure. This finding agreed with the enhancement of the milk clotting activity by the polyethyleneglycol. The method appears to be suitable as a first step for the purification of these proteins from their natural sources.     

The application of aqueous two-phase systems to the purification of pharmaceutical proteins from transgenic sheep milk

Transgenic sheep milk containing the protein human₁-Antitrypsin (AAT) was partitioned in Poly(ethyleneglycol) (PEG)-Sulphate and PEG-Phosphate biphasic systems. Individual partition coefficients for AAT and some of the milk proteins were determined in these systems. The effects of PEG molecular weight, pH and the inclusion of NaCl on the partitioning of the proteins were also studied. It was found that increasing the concentration of NaCl and decreasing the molecular weight of the PEG resulted in an increase of the partition coefficients of the proteins to the upper (PEG) phase. This partitioning effect was greater for the more hydrophobic proteins and particularly in systems having a pH close to the isoelectric point of the protein. Solubilities of the proteins in increasing concentrations of ammonium sulphate were measured in order to investigate the effects of hydrophobic and electrostatic interactions on the partitioning of these proteins in aqueous two-phase systems. Those proteins that precipitated at low levels of ammonium sulphate showed an increase in partition coefficient at low concentrations of NaCl, or they were precipitated at the interface of the phases at low concentrations of NaCl. Proteins that had low salting out constants in ammonium sulphate solutions were relatively unaffected by NaCl in ATPS. It is probable however that conformational changes and the state of aggregation of proteins are also important and should be invoked in describing the partitioning behavior observed for -Lg for example. Comparison of theoretical and experimental values for AAT yield and purity showed clearly that partition coefficients are influenced by the degree of purity and values obtained with purified standards are not necessarily the same as for the same protein present in a complex mixture. Under the most favourable conditions using a 4% w/w loading of transgenic ovine milk, we obtained a 91% yield of AAT in the PEG phase with a purity of 73%.This revised version was published online in October 2005 with corrections to the Cover Date.     

Affinity gel electrophoresis as a predictive technique in the fractionation of transgenic sheep milk proteins by affinity aqueous two-phase partitioning

Affinity electrophoresis in the presence of various triazine dyes of sheep milk proteins, including transgenically-introduced human ₁-antitrypsin, has been evaluated as a predictive technique for possible large scale affinity-driven aqueous two-phase purifications. The success of the approach suggested it has potential as a general method for the rapid screening of ligands using only g amounts of sample, that could be applied to many complex mixtures before embarking on more costly and time-consuming two-phase partitioning experiments.     

Study of the serum albumin-polyethyleneglycol interaction to predict the protein partitioning in aqueous two-phase systems

The theoretical framework based only on the excluded volume forces is not enough to explain the bovine serum albumin partitioning behaviour in aqueous biphasic systems. The goal of this work is to look at the phase separation via the polymer effect on the water structure. Our findings suggest that polyethyleneglycol 600-protein interaction is conducted by van der Waals forces between the hydrophobic surfaces from PEG and protein molecules, which implies the rupture of hydrogen bonds from the structured water in their neighbours. Therefore, the protein will concentrate in the most water-structured phase (polyethyleneglycol) in order to reach the minimal free energy condition. When polyethyleneglycol molecular weight increases, its exclusion from protein surface prevails, thus pushing the bovine serum albumin to the bottom phase.