A crystallization technique for obtaining large protein crystals with increased mechanical stability using agarose gel combined with a stirring technique
Introduction
Determination of the three-dimensional (3D) structures of protein molecules is crucial for structure-guided drug design and controlled drug delivery. X-ray diffraction studies and neutron diffraction studies are widely used to determine the 3D structures of protein molecules, but cannot be applied to crystals of insufficient size or quality. Therefore, the difficulty of generating large high-quality protein crystals remains a bottleneck in the field of molecular structure study. In addition, the fragility of protein crystals limits the success rate of structure analysis, because fragile crystals can be damaged during the preparation process before X-ray or neutron diffraction analysis. To overcome these problems, there is need of a crystallization technique that achieves protein crystals of sufficient quality, size and physical strength.
Many crystallization techniques have been investigated, including the batch method, vapor diffusion method [1], the counter-diffusion method [2], the microseeding method [3], crystallization in hydrogel [4], [5], [6], [7], [8], [9], [10], or crystallization under microgravity [11], [12], [13], a magnetic field [14], [15], or forced solution flow [10], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28]. When using these protein crystallization techniques, controlling the solution flow is one of the most important parameters [17], [18], [24], [29], and several groups have developed practical techniques for this purpose.
Establishing further practical and effective crystallization techniques, we have to get protein crystals to overcome handling damages before X-ray diffraction experiment including the soaking process using cryo-protectant solutions and radiation damage during X-ray diffraction experiment. Crystallization techniques using hydrogel are useful to overcome these problems. Among typical gels used for protein crystallization, agarose gels are the most employed hydrogels in protein crystallization because of its stability, easy for use and high familiarity for biochemists [6]. Crystallization using agarose gel has some advantages; the nucleation enhancement effect [7], [30], the impurity filtering effect during crystal growth [31], the suppression of heat convective transport and crystal sedimentation [32], and the enhancement of crystal strength by incorporation of agarose gel fibers into protein crystals [6], [8]. Especially the enhancement of crystal strength by agarose gel has great advantages for overcoming handling damage before X-ray diffraction studies or osmotic shock damage during ligand soaking experiments [8], [9]. In fact many crystals including model proteins had been improved by the agarose gel crystallization techniques [8], [33]. Contrary to the very useful aspects, the batch method combined with agarose gel occasionally increases nucleation probability too much, as a result problems such as size degradation of each crystals and/or generation of polycrystals occur. The agarose gel method combined with the counter diffusion method is one of a useful technique to get fine crystals, though there arise some crystal size variation due to the gradation of protein and precipitant concentration [34]. In this study, resolving the problem that too much nucleation and the size degradation, we combined the hydrogel crystallization with the stirring technique [35], [36], [37] which enables us to decrease the nucleation probability and increase the size of each crystal. Gel existence must inhibit solution convection and forced solution flow as it reported [32]. However, from the view point of the pore size of agarose gel, it is enough larger than protein single molecules [38], [39] and that is why protein crystals can grow in high concentrated agarose gel media. Considering the relation between the agarose gel pore size and protein molecule size, we thought that there is possibility to affect the protein molecules in agarose gelled solution by stirring operation. To test the effect of the stirring technique on gelled solution, we used the findings that appropriate solution stirring made delay of nucleation and the number of crystals was finally lowered. Using this newly developed crystallization technique incorporating the stirring of a gel solution, we achieved effects comparable to those from either of the previous methods used singly.
Section snippets
Materials
Three-times recrystallized hen egg-white lysozyme (HEWL) from Wako Co. (Catalog no. 120-02674) was used without further purification. Sodium chloride and sodium acetate were purchased from Wako Co., Japan (Catalog nos. 192-13925 and 192-01075, respectively). Agarose was purchased from Wako (Catalog no. 50101). The gelling temperature of the agarose was 299–303 K. We used 1 ml glass vials for crystallization. The 0.2 μm pore size filters were purchased from Advantec (Catalog no. 25CS020AS).
Crystallization
Six
Effects of the agarose gel concentration and the rotating speed on the crystal nucleation.
Fig. 2(a) shows the relation between the number of crystals and the rotating speed just 5 days after the crystallization started. The agarose gel concentrations of 0%, 0.5%, 1.0% and 2.0% were tested, and the results are shown in the figure using blue, green, yellow and red diamonds, respectively. Under the condition using 0% agarose gel, crystal nucleation did not occur at any of the rotating speeds within five days after crystallization, except in one vial at 0 rpm (blue diamonds). Thus the
Conclusion
We suggested protein crystallization technique in 0.0–2.0% agarose gel solution with the stirring operation, and investigated the nucleation probability, the growth rate of the crystals. By increasing the agarose gel concentration from 0% to 2.0%, the nucleation probability monotonically increased. Increasing the rotating speed under all of the agarose gel concentrations used decreased the number of crystals. Under the condition using 0.5% agarose gel, we measured the crystal size and growth
Acknowledgment
We acknowledge Mr. Ryota Mori for his experimental support. This work was supported in part by the Osaka University Program for the Support of Networking among Present and Future Researchers. We also acknowledge funding in the form of a JSAP KAKENHI Grant-in-Aid for Scientific Research B (Grant no. 23360011) to Y.M.
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