The crystallization of biomacromolecules by using membrane-assisted crystallization technology is described in this chapter. The discussion will focus on the interest in growing protein crystals and the current difficulties in obtaining them by conventional crystallization methods, the possibility to use MAC for the growth of protein crystals and the influence of controlling the transmembrane flux on crystallization kinetics, the function of polymeric membranes as solid support for heterogeneous nucleation and the correlation between membrane physical properties and the energetics of nucleation, and the effect of forced solution-flow convection on crystallization kinetics and its influence on crystal properties.
Interest in Protein CrystallizationThe functions of biomolecules are strictly connected to their three-dimensional molecular structures. Therefore, the determination of the spatial arrangement of atoms or groups of atoms in protein molecules is of fundamental importance for the understanding of their biological activity and for designing new drug molecules that can act at active sites. 1 The 3D structure of proteins can be determined by X-ray or neutron diffraction and nuclear magnetic resonance (NMR) spectroscopy. However, the crystallographic method is the one of choice, especially for molecules that are too large (M.W. > 20 kDa). Crystallography gives a higher resolution, reaching less than 1 Å for particularly complex systems like membrane-protein or viruses. 2 The basic requisite for structure resolution at the atomic level by X-ray diffraction is the availability of well-diffracting crystals of adequate size. 3 193 Membrane-Assisted Crystallization Technology Downloaded from www.worldscientific.com by CHINESE UNIVERSITY OF HONG KONG on 10/09/15. For personal use only.194 Membrane-Assisted Crystallization TechnologyAmong proteins, enzymes are the most efficient known catalysts because of their high substrate specificity. 4 However, their systematic utilization in solution at industrial scale has been rather limited due to their intrinsic labile nature under operative conditions. Poor chemical and mechanical stability at extreme temperatures and pH, high pressure, mechanical stress, and the presence of denaturing solvents and other chemicals, are the factors that have prevented enzymes from being extensively used. Also, in the solid state, proteins are normally characterized by pronounced fragility or shattering under relatively mild conditions. On the other hand, the possibility of producing a large amount of protein crystal with reduced, uniform, and predictable size, slow dissolution kinetics, and high chemical and mechanical stability would have remarkable implications in many biotechnological applications, e.g. for sustained constant rate drug release, in environmental protection applications, in chemical synthesis, etc. 5 In this respect, cross-linked enzyme crystals (CLECs) 6,7 are interesting systems whose production is a relatively simple, fast, and less expensive technique and one that does ...