Containerless protein crystallization in floating drops: Application to crystal growth monitoring under reduced nucleation conditions

A micromethod was developed for the batch crystallization of proteins under conditions were the solution has no contact with the container walls. Drops of crystallization solutions (5 to 100 mu l) are placed at the interface between two layers of inert and non-miscible silicone fluids contained in square glass or plastic cuvettes. The densities of the fluids are either lower or higher than those of the major precipitating agents of macromolecules, including aqueous solutions containing salts, polyethylene glycols or alcohols. Several proteins and a spherical plant virus were crystallized in the temperature range 4 degrees C-20 degrees C using this set-up. A thermostated device was built for the dynamic control of the temperature of crystallization drops and the monitoring of crystal growth by video-microscopy. In all cases, the habit of the crystals grown ia floating drops are identical to those of controls grown in sealed glass tubes without silicone fluid. The comparison of the number of crystals in drops kept under one layer of fluid and in floating drops of the same volume indicates that heterogeneous nucleation is minimized when protein crystallization is performed in floating drops. The advantages and limitations of this novel containerless crystallization method are discussed.