C. J. Dale

Generic application of an aqueous two-phase process for protein recovery from animal blood

The recovery of proteins from animal blood using an established two-stage extraction process was selected as a practical model system to study the generic application of polyethylene glycol (PEG)-phosphate aqueous two-phase systems (ATPS). Processing of whole bovine blood in the ATPS two-stage process resulted in the partition of soluble protein (e.g. bovine serum albumin (BSA), haemoglobin, IgG; partition coefficient K=55) into a PEG-rich top phase and cell debris into a phosphate-rich bottom phase. Subsequent back extraction of soluble protein into a second phosphate-rich bottom phase resulted in a maximum overall protein recovery of 62%. The increased protein concentration within the ATPS (from 1.2 to 7.0 mg/g) caused a decreased in the recovery to 44%. Recycling of PEG into the initial extraction stage did not significantly influence the partition behaviour of protein over the equivalent of 20 operational cycles, but protein recovery decreased from 44 to 37%. The extreme conditions (waste material characterised by the presence of solids and impurities) in which the implementation of this ATPS process was tested, confirms the potential for the generic application of ATPS for processing complex biological suspensions to achieve a simple primary recovery and partial purification of target protein solutes.

Aqueous two-phase partition of complex protein feedstocks derived from brain tissue homogenates

This study describes the application of aqueous two-phase partition using polyethylene glycol (PEG)-potassium phosphate systems for the direct recovery of proteins, and aggregates thereof, from mammalian brain tissue homogenates. Investigation of established methodologies for the purification of prion proteins (PrP) from bovine brain affected with transmissible spongiform encephalopathy (BSE) has identified an alternative purification regime based on aqueous two-phase partition. This circumvents energy-intensive and rate-limiting unit operations of ultracentrifugation conventionally used for isolation of PrP. Selectivity of various PEG-phosphate systems varied inversely with polymer molecular mass. The maximum protein recovery from bovine brain extracts was obtained with systems containing PEG 300. Manipulation of the aqueous environment, to back-extract protein product from the PEG-rich top phase into the phosphate-rich lower phase, enabled integration of ATPS with conventional hydrophobic interaction chromatography (HIC) which selectively removes obdurate contaminating proteins (i.e. ferritin).

Studies on the physical and compositional changes in collapsing beer foam

Abstract Batches of foam were characterised by analysis of conductivity and quantitative changes in mass and solute composition of material retained both within foam and foam drainage. The conductivity of dispensed beer foam declined exponentially during the initial stages of foam collapse, followed by a secondary or consolidation stage characterised by deviation from exponential decay and increases in the concentration of polypeptide material in both foam and foam drainage. Analysis of the amino acid composition of whole collapsed foams suggested that polypeptides of enhanced hydrophobicity were selectively partitioned to the gas–liquid interface at foam formation and subsequent consolidation. Estimates of the changes in total mass of the liquid phase within dispensed beer foam suggested that foam collapse proceeded by two distinct stages of exponential decay characterised by different rates ( k 1 , k 2 ). The first rate ( k 1 ) corresponded to the initial stage of foam collapse and accounted for 85–90% loss of foam mass. This was followed by a slower rate of collapse ( k 2 ) which corresponded to the consolidation stage. The results from physical and compositional analyses suggest that the initial stage of foam collapse is dominated by gravitational drainage from a liquid rich foam followed by a change in emphasis to coalescence and bubble rupture during the consolidation and residual stages of foam collapse. These findings contribute to the understanding and characterisation of foam formation and stability.

Aqueous two-phase fractionation of biological suspensions for protein recovery from bovine blood

The practical application of a two-stage aqueous two-phase systems (ATPS) for the fractionation and recovery of proteins from biological suspensions is described. A model process for the recovery of proteins from whole bovine blood was selected to study the implementation of an ATPS process. Recycling of used PEG into the initial extraction stage did not significantly influence the partition behaviour of serum albumin in subsequent cycles.