Effects of Polyethylene Glycol 4000 and Sodium Chloride: 3-dimensional phase diagram for a protein crystallization

S. Takahashi¹, B. Yan¹, N. Furubayashi², K. Inaka², H. Tanaka¹

¹Confocal Science Inc., 2-12-2 Iwamoto-cho, Chiyoda-ku, Tokyo 101-0032 JAPAN

²Maruwa Foods and Bioscience Inc., 170-1 Tsutsui-cho, Yamatokoriyama, Nara 639-1123 JAPAN

tanakah@confsci.co.jp

Crystallization is associated with a decrease in the solvent accessible surface of a protein. For the crystallization of protein molecule, it is important to neutralize its charge with counterions to produce electroneutrality [1]. Previously, we have shown that the importance of co-existing sodium chloride in polyethylene glycol (PEG) 4000 - based crystallization solution, the most widely used precipitant [2]. However, the relation between PEG 4000 and sodium chloride could not be enough demonstrated. Therefore, we performed lysozyme crystallization in the concentration range of PEG 4000 (0-25%) and sodium chloride (0.1-1.0M) using batch method in a capillary of 0.3mm diameter to make a 3-dimensional phase diagram. In the course of experiments, we found that lysozyme crystals could grow in lower concentration (0.1~0.2M) of sodium chloride if PEG 4000 was co-existed. In the solution of lower PEG 4000 concentration, a few but large crystals grew (Fig. 1-1). On the other hand, in the solution of higher PEG 4000 concentration, many but small crystals grew (Fig. 1-2), and if the PEG 4000 concentration is much higher, large but clustered crystals grew (Fig. 1-3). These results are consistent with the classical nucleation theory [3, 4, 5]. The X-ray diffraction data showed that the crystals grown in higher PEG 4000 solution diffracted to higher resolution. These results suggest that it may be better to use solution of lower concentration PEG with certain amount of sodium chloride to grow single crystals, but optimization of the concentration of PEG 4000 and sodium chloride may be a critical problem from a point of view of diffraction study.

Figure 1-1. Lysozyme crystals grown in 5% PEG 4000 and 0.2M NaCl

Figure 1-2. Lysozyme crystals grown in 15% PEG 4000 and 0.2M NaCl

Figure 1-3. Lysozyme crystals grown in 20% PEG 4000 and 0.1M NaCl

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