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Studies on the partition and purification of penicillin acylase from osmotic shock extract Escherichia coli were performed in poly (ethylene glycol)-citrate systems. Both partition behavior of the enzyme and total protein are similar to those described in other reports increasing ,with pH and tie-line length and decreasing with PEG molecular weight. However, some selectivity could be attained with PEG 1000 systems and long tie-line at pH 6.9. In these conditions 2.6 fold purification with 83% yield were achieved. Influence of pH on partition shows that is the composition of the system and not the net charge of the enzyme that determine the behaviour in these conditions. Addition of NaCl to PEG 3350 systems significantly increases the partition of the enzyme. Althought protein partition also increased, purification conditions were possible with 1.5 M NaCl where 5.7 fold purification and 85% yield was obtained. This was possible due to the higher hydrophobicity of the enzyme compared to that of most of contaminants proteins

Phase separation has long been observed within aqueous mixtures of two or more different compounds such as proteins, salts, polysaccharides and synthetic polymers. A growing body of experimental evidence indicates that phase separation also takes place inside living cells, where intrinsically disordered proteins and other molecules such as RNA are thought to assemble into membraneless organelles. These structures represent a new paradigm of intracellular organisation and compartmentalisation in which biochemical processes can be coordinated in space and time. Two thermodynamic driving forces have been proposed for phase separation: the strengths of macromolecule-macromolecule and macromolecule-H2O interactions, and the perturbation of H2O structure about different macromolecules. In this Perspective, we propose that both driving forces act in a concerted manner to promote phase separation, which we describe in the context of the well-known structural dynamics of intrinsically disordered proteins in the cellular milieu. We further suggest that this effect can be extended to explain how the partial unfolding of globular proteins can lead to intracellular phase separation.

The current study explores the possibility of using a polyethyleneglycol(PEG)-ammonium sulphate aqueous two-phase system (ATPS) as an early step in a process for the purification of a model 6.1 kbp plasmid DNA (pDNA) vector. Neutralised alkaline lysates were fed directly to ATPS. Conditions were selected to direct pDNA towards the salt-rich bottom phase, so that this stream could be subsequently processed by hydrophobic interaction chromatography (HIC). Screening of the best conditions for ATPS extraction was performed using three PEG molecular weights (300, 400 and 600) and varying the tie-line length, phase volume ratio and lysate load. For a 20% (w/w) lysate load, the best results were obtained with PEG 600 using the shortest tie-line (38.16%, w/w). By further manipulating the system composition along this tie-line in order to obtain a top/bottom phase volume ratio of 9.3 (35%, w/w PEG 600, 6%, w/w NH4)2 SO4), it was possible to recover 100% of pDNA in the bottom phase with a three-fold increase in concentration. Further increase in the lysate load up to 40% (w/w) with this system resulted in a eight-fold increase in pDNA concentration, but with a yield loss of 15%. The ATPS extraction was integrated with HIC and the overall process compared with a previously defined process that uses sequential precipitations with iso-propanol and ammonium sulphate prior to HIC. Although the final yield is lower in the ATPS-based process the purity grade of the final pDNA product is higher. This shows that it is possible to substitute the time-consuming two-step precipitation procedure by a simple ATPS extraction.

Penicillin acylase purification from an Escherichia coli crude extract using PEG 3350sodium citrate aqueous two phase systems was optimized. An experimental design was used to evaluate the influence of PEG, sodium citrate and sodium chloride on the purification parameters. A central composite design was defined centred on the previously found conditions for highest purification from an osmotic shock extract. Mathematical models for the partition coefficient of protein and enzyme, balance of protein and enzyme, yield and purification were calculated and statistically validated. Analysis of the contours of constant response as a function of PEG and sodium citrate concentrations for three different concentrations of NaCl revealed different effects of the three factors on the studied parameters. A maximum purification factor of 6.5 was predicted for PEG 3350, Sodium Citrate and NaCl concentrations of 15.1%, 11.0% and 8.52% respectively. However under these conditions the predicted yield was 61%. A better compromise between these two parameters can be found by superimposing the contour plots of the purification factor and yield for 10.3% NaCl. A region in the experimental space can be defined where the purification factor is always higher than 5.5 with yields exceeding 80%.

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