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The optimization of solution flow rate producing high-quality and large protein crystals

Yuki Hayashi 1Mihoko Maruyama 1Satoshi Nakayama 1Yoshinori Takahashi 1Hiroshi Yoshikawa 2Masashi Yoshimura 1Masaru Tachibana 3Haruhiko Koizumi 4Sigeru Sugiyama 5Hiroaki Adachi 1,6Kazufumi Takano 6,7Satoshi Murakami 6,8Hiroyoshi Matsumura 1,6Tsuyoshi Inoue 1,6Yusuke Mori 1,6

1. Graduated School of Engineering, Osaka University (OSAKAUNIV), Osaka, Japan
2. Saitama University, 255 Shimo-ookubo, Sakuraku, Saitama 338-8570, Japan
3. Graduate School of Nanobioscience, Yokohama City University, Yokohama 236-0027, Japan
4. Institute for Materials Research, Tohoku University, Sendai 980-8577, Japan
5. Graduate School of Science, Osaka University, Osaka 565-0871, Japan
6. SOSHO Inc., Osaka 565-0871, Japan
7. Kyoto Prefectural University, Kyoto 606-0823, Japan
8. Tokyo Institute of Technology, Tokyo 152-8550, Japan

 Large protein crystals with high-quality are essential for determining the three-dimensional structure of proteins by X-ray diffraction measurement and neutron diffraction measurement. We have developed the top-seeded solution growth with the floating and solution-stirring technique (TSSG-FAST)1 and succeeded to grow crystals of HIV-1 protease suitable for neutron diffraction measurement (Fig. 1). Using these crystals, structual information about the H atoms of HIV-1 protease was successfully determined. TSSG-FAST is an efficient technique for producing high-quality and large protein crystals. To make the technique highly-developed, we studied the correlation of the growth rate and the quality with the solution flow rate, and decided the best flow rate for growing high-quality and large crystals of proteins.___9.jpgFig. 1 Schematic illustration of the crystallization setup for TSSG-FASTFig. 2 Schematic illustration of the HEWL and the morphology of spiral growth hillock

 Crystals of hen egg-white lysozyme (HEWL) were grown by the TSSG-FAST under various solution flow rates, which were determined by flow analysis. Growth rate of (110) face was obtained by observing the lengths along [110] direction at the starting and the ending day (Fig. 2). We also observed the morphology of spiral growth hillocks on {110} faces by laser confocal microscopy combined with differential interference contrast microscopy (LCM-DIM). The morphology is affected by impurities and the aspect ratio (long to short axes) of steps can be used as a measure of impurity effects (Fig. 2).2 At the flow rate of 0 μm/sec, growth rate of (110) face was 0.2 μm/h and the aspect ratio of long to short axes was 4.1 ± 0.3 (Fig. 3). With increasing the flow rate, the both increased and had the maximum at the flow rate of 140 μm/sec, and then decreased. This result showed that there exists the best solution flow range, and solution flow faster than 140 μm/sec is not suitable for protein crystal growth. We consider that this result can be explained by the competition between the mass transfer of solutes and the inhibition effect of the impurities.3
Crystal defects (especially dislocations) in the HEWL crystal grown by the TSSG-FAST at the flow rate of 8 μm/sec were investigated by synchrotron monochromatic-beam X-ray topography (Fig. 4). The dislocation density of the crystal grown by TSSG-FAST was 101 ~ 102 cm-2, which was lower than the dislocation density of a crystal grown by non-stirring technique.4  This result shows that the TSSG-FAST have high possibility to reduce dislocations in crystals. As a result, we suggest that the TSSG-FAST can produce high-quality and large crystal of protein, especially at the flow rate lower than 140 μm/sec.

Fig. 3 Aspect ratio and Growth rates of (110) face as a function of the flow rate

Fig. 4 Synchrotron monochromatic-beam X-ray topography (a)without stirring (b)TSSG-FAST

1) N.Shimizu, et al., Cryst. Growth Des. 10 (2010) 2990.
2) P. Dold, et al., J. Cryst. Growth 293 (2006) 102.
3) M. Maruyama, et al., Cryst. Growth Des. 12 (2012) 2856.
4) Y. Mukobayashi, et al., Phys. Status Solidi A206 (2009) 1825.


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Presentation: Oral at 17th International Conference on Crystal Growth and Epitaxy - ICCGE-17, General Session 3, by Yuki Hayashi
See On-line Journal of 17th International Conference on Crystal Growth and Epitaxy - ICCGE-17

Submitted: 2013-04-05 07:04
Revised:   2013-07-16 16:59