Controlled Nucleation and Growth of Protein Crystals by Solvent Freeze-Out

Solvent freeze-out technology has been developed as a new concept in the field of protein crystallization. This technology allows the separation of the nucleation and growth steps but requires an understanding of the thermodynamics of the complex mixture of protein, solvent, salt, and buffer at temperatures for which few accurate data currently exist. The phase diagram of the given protein system was systematically investigated and confirmed for the identification of optimal crystallization conditions for zone I, which is the best region of protein crystallization, by employing a preliminary screening. As an initial necessity for protein crystallization, the delicate balance between repulsive and attractive forces in the given protein system was found at pH 4.4 and 5 wt % NaCl. The precise value of the supersaturation level (S) of zone I was estimated to be 9.56 ≤ S ≤ 29.34 after a statistical analysis of the initial screening by a Linbro test. Next, the supersaturation levels for the metastable zone were identified to be 7.0 ≤ S < 17.1 from a statistical analysis of all of the experimental results from both the Linbro test and the individual crystal growth measurements. Protein crystallization in zone I by the freeze-out technology was carried out and evaluated. The key process variable levels (KPVLs) were operated within the boundary of the phase diagram that was confirmed by the preliminary screening. With a NaCl concentration of 5 wt % at pH 4.4, quite a good quality of tetragonal hen egg-white lysozyme (HEWL) crystals was produced as a result of proper tuning of the net surface charge of HEWL, even with a very low value of the initial protein concentration. The number of tetragonal HEWL crystals was increased by increasing the ice mass, since the cooling rate applied to the system determines the ice growth rate and therefore the nucleation and growth rates of the protein crystals. Hence, nucleation of the given protein system can be controlled by moderate adjustment of the ice growth rate.