Cryst. Res. Technol. 41, No. 11, 1063 – 1066 (2006) / DOI 10.1002/crat.200610722 © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Sedimentation as a tool for crystallization from protein mixtures I. Dimitrov and C. N. Nanev* Rostislaw Kaischew Institute of Physical Chemistry, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria Received 7 June 2006, accepted 25 June 2006 Published online 1 October 2006 Key words protein crystallization, apoferritin, lysozyme, zonal sedimentation, free interface diffusion. PACS 87.14.Ee, 87.15.Nn Crystals from apoferritin which is an iron-free form of protein ferritin were obtained from protein mixtures lysozyme/apoferritin using sedimentation under high gravity. Solution containing apoferritin at concentration as high as 5mg/ml in the presence of 25mg/ml lysozyme and overlaid on 5%(w/v) CdSO 4 in 0,2M/L NaAC, pH=5 still favors apoferritin crystal formation under normal gravity conditions, but at apoferritin concentrations <0,5mg/ml (~1,14µM/L) in 25mg/ml (~1,71mM/L) lysozyme only the sedimentation in a centrifuge appears to be useful for separating the apoferritin molecules from the mixture followed by apoferritin crystallization in the same system. The very high molecule number ratio (~1:10 3 ) of two proteins is used to stress on the observed effect. © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 1 Introduction One of the main goals of protein crystallization is the obtaining of relatively large protein crystals of good quality, which is the most important stage in the process of protein structure determination via X-ray diffraction. Although this is a tough task more and more proteins are being crystallized successfully and have theirs structures deposited at the Protein Data Bank. Number of methods have already been developed or have been accommodated for the investigation of protein crystallization [1-3]. Sedimentation under high gravity is powerful physicochemical method [4, 5] and certain research has been done in crystallization by means of it [6-8, 9]. It is well known that protein purification is a multistage and difficult process and the purity that can be achieved is commonly around 95%. Since the protein solutions used for crystallization are sensitive to impurity content [10, 11] which may comprise dust particles, another protein or even conformational heterogeneity between molecules of one protein, we suggest a way for obtaining of protein crystals from protein mixtures. In our experiment we have used deliberately mixed solutions at different concentration ratios of two otherwise readily crystallizing proteins – chicken egg-white lysozyme and apoferritin from horse spleen. 2 Experimental set-up Chicken egg-white lysozyme was purchased from Sigma-Aldrich Co. Apoferritin from horse spleen (Sigma Chemicals Co.) was used additionally purified by FPLC as described in [12]. The experimental set-up is shown at figure 1. Glass capillary tubes fused at one end were filled with solution (52µl) of 5%w/v CdSO 4 in 0,2M/L NaAC buffer, pH=5. A small volume (4µl) of protein mixture lysozyme toward apoferritin in molar ratio 1500:1 (the largest one) was carefully overlaid at the top of the higher density salt solution. Bidistilled water was used as a solvent for the proteins. The capillaries were then centrifuged at 2200g for 5 hours. The use of centrifugation avoids the need for additional preliminary filtration of protein solutions. Dust particles as well as apoferritin dimers or high order protein oligomers are separated from the protein mixture ____________________ * Corresponding author: e-mail: nanev@ipchp.ipc.bas.bg