A simple technique is presented for non-chromatographic purification of recombinant proteins expressed in Escherichia coli. This method is based on a reversibly precipitating, self-cleaving purification tag. The tag is made up of two components: an elastin-like polypeptide (ELP), which reversibly self-associates in high-salt buffers at temperatures above 30 degrees C; and an intein, which causes the ELP tag to self-cleave in response to a mild pH shift. Thus, a tripartite ELP-intein-target protein precursor can be purified by cycles of salt addition, heating and centrifugation. Once purified, intein-mediated self-cleavage, followed by precipitation of the cleaved ELP tag, allows easy and effective isolation of the pure, native target protein without the need for chromatographic separations. Recoveries of 50-100 mg of cleaved, native target protein per liter of shake-flask culture have been achieved for over a dozen proteins, typically in 8-24 h depending on specific process parameters.

Observing the phase separation of deeply quenched, low viscosity liquid mixtures we inferred that the process is driven by the convection due to capillary forces, and not by molecular diffusion neither by gravity, heat or surface effects. After quenching a partially miscible, initially homogeneous, off-critical liquid mixture to a temperature T deeply below its critical point of miscibility Tc, with |T−Tc|/Tc≈0.1, we observed the formation of rapidly coalescing droplets of the minority phase, whose size grows linearly with time. Following the motion of isolated 10 μm droplets, we saw that they move in random directions at speeds exceeding 100 μm/s, showing that during most of the process the system is far from local equilibrium. Eventually, when their size reaches the capillary length, the nucleating drops start sedimenting as gravity becomes the dominant force. This behavior was observed for both density-segregated and density-matched systems, irrespectively whether they were kept in horizontal or vertical cells. The experiments were repeated using both untreated (i.e., hydrophilic) and modified (i.e., hydrophobic) cell walls, with identical results and, in addition, no bulk motion was observed when the mixture was replaced with water, showing that the observed convection is not induced by gravity, neither by surface or temperature effects. Using a simple dimensional analysis of the governing equations based on the diffuse interface model, we showed that convection is induced by the coalescence among drops which, in turn, is the result of a nonequilibrium capillary force that indeed dominates both diffusion and gravity forces.

After quenching a partially miscible, initially homogeneous, critical liquid mixture to a temperature T deeply below its critical point of miscibility, we observed the formation of rapidly coalescing droplets, whose size grows linearly with time, thus indicating that the phase separation process is driven by convection. Eventually, when their size reaches a critical length, which is roughly equal to one-tenth of the capillary length, the nucleating drops start sedimenting and the two phases rapidly segregate by gravity. This behavior was observed for both densitysegregated and quasi-isopycnic systems, showing that gravity cannot be the driving force responsible for the enhancement of the coalescence among the nucleating drops. This result is in line with previous theoretical works based on the diffuse interface model, predicting that the phase separation of low-viscosity liquid mixtures is a convection-driven process, induced by a body force which is proportional to the chemical potential gradients. Finally, at later times, following the evolution of isolated drops of the secondary emulsion, we saw that their size grows in time like t 1/3 . † "A wooden boy?!" (R. Shinnar, freely quoted from Pinocchio, by Collodi, 1864, via Walt Disney).

Complete phase segregation may occur on a 10 cm scale even in the absence of buoyancy due to unidirectional, large-scale rapid bulk flow. Using a hexadecane-acetone nearly density-matched liquid mixture in a 20-cm-long condenser tube with a 1 cm diameter, we observed the rapid axial migration of the acetone-rich drops towards the warmer regions of the condenser. Conversely, the hexadecane-rich drops moved in the opposite direction, therefore ruling out thermocapillary effects as a possible explanation of the phenomenon. These flows lead to a complete phase segregation within 10 s, with the formation of a single interface perpendicular to the axial direction. Changing the temperature gradient along the tube from 0.25 to 1°C/cm no change was detected, with typical drop speeds up to 6 cm/ s, irrespective of the distance of the drop from the wall, showing that the phenomenon is not due to a flow instability.