Membrane crystallization of lysozyme under forced solution flow

The production of protein crystals with adequate size and high lattice perfection for X-ray analysis appears as the bottleneck for obtaining diffraction data at a resolution sufficient to establish 3D structure-function correlation. In previous investigations, microporous hydrophobic membranes have emerged as effective tools for macromolecular crystallization: polymeric supports have been used both to generate a controlled supersaturated environment, and to promote heterogeneous nucleation. In this work, experiments have been carried out under forced convection in order to verify if the laminar flow of the mother solution through polypropylene hollow-fibers can induce an ordered growth of hen egg white lysozyme (HEWL) crystals. In the range of flow velocities investigated (210-2100 μm/s), the values of the kinetic Peclet number confirmed a shifting of the operative system point to a regime with significantly faster transport and higher relative weight of interfacial kinetics in the overall control of the crystallization process. Morphological analysis proved that tetragonal lysozyme crystals became convectively-oriented, aligning their c-axis in the flow direction. Kinetic analysis evidenced a maximum in crystal growth rate in correspondence of a flow velocity of about 1100 μm/s for crystallization tests performed at NaCl 3% (w/v), MgCl2 10% (w/v), and T 5 °C. The enzymatic activity of the crystallized product excluded denaturation of the recirculating protein solution. Finally, crystals have been qualitatively assessed by X-ray crystallographic analysis carried out on a rotating anode X-ray generator (Cu Kα, λ = 1.5418 Å) operating at 45 kV and 100 mA.