Numerical analysis of the depletion zone formation around a growing protein crystal.

It is expected that a protein depletion zone and an impurity depletion zone are formed around a crystal during protein crystal growth if the diffusion field around the crystal is not disturbed. The growth rate of the crystal may be decreased and the impurity uptake may be suppressed to result in highly ordered crystals if these zones are not disturbed. It is well known that a microgravity environment can reduce convective fluid motion, and this is thought to disturb the depletion zones. Therefore, we expect that crystals grown in space can attain better quality than those grown on the ground. In this study, we estimate the depletion zone formation numerically and discuss the results of crystallization in space experiments. In case of alpha-amylase, most of the crystals form a cluster-like morphology on the ground using PEG 8000 as a precipitant. However, in space, we have obtained a single and high-quality crystal grown from the same sample compositions. We have measured the viscosity of the solution, the diffusion coefficient, and the growth rate of protein crystals on the ground. Applying numerical analysis to these values a significant depletion zone was expected to form mainly due to higher values of the viscosity. This might be one of the main reasons for better quality single crystals grown in space, where the depletion zone is thought to remain undisturbed. For protein crystallization experiments, salts are widely used as a precipitant. However, in that case, reduced concentration depletion zone effects can be expected because of a low viscosity. Therefore, if it is possible to increase the viscosity of the protein solution by means of an additive, the depletion zone formation effect would be enhanced to provide a technique that would be especially effective in space.

A simplified counter diffusion method combined with a 1D simulation program for optimizing crystallization conditions.

We developed a new protein crystallization method has been developed using a simplified counter-diffusion method for optimizing crystallization condition. It is composed of only a single capillary, the gel in the silicon tube and the screw-top test tube, which are readily available in the laboratory. The one capillary can continuously scan a wide range of crystallization conditions (combination of the concentrations of the precipitant and the protein) unless crystallization occurs, which means that it corresponds to many drops in the vapor-diffusion method. The amount of the precipitant and the protein solutions can be much less than in conventional methods. In this study, lysozyme and alpha-amylase were used as model proteins for demonstrating the efficiency of this method. In addition, one-dimensional (1-D) simulations of the crystal growth were performed based on the 1-D diffusion model. The optimized conditions can be applied to the initial crystallization conditions for both other counter-diffusion methods with the Granada Crystallization Box (GCB) and for the vapor-diffusion method after some modification.