Narrow size distribution of lysozyme crystals in a reverse vapor diffusion set-up

Abstract

The reverse vapor diffusion method is among the rarest methods used in protein crystallization studies. In the common case, it is accompanied by a decrease in the supersaturation of the crystallization system and thus by a decrease in the probability of nucleation and successive overall crystallization. We investigated the crystallization of lysozyme in supersaturated solutions at 33 °C, where small volumes of water gradually and completely evaporated and gently condensed in the crystallization solution, defining the final composition of the crystallization system. The results showed that the reverse vapor diffusion method could be successfully used to obtain crystal populations with narrow size distribution in the following range of final protein and precipitant concentrations: 28.6 mg ml⁻¹ lysozyme, 3.57% (w/v) NaCl – 25 mg ml⁻¹ lysozyme, and 3.13% (w/v) NaCl. Our hypothesis is that water condensation forces the crystallization system out of the nucleation zone much faster than crystal nucleation and growth. The process depends predominantly on the volume of the water phase and temperature.