science24.com - The optimization of solution flow rate producing high-quality and large protein crystals

Search for content and authors

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

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

Abstract

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.

Fig. 1　Schematic illustration of the crystallization setup for TSSG-FAST

Fig. 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

Reference
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.

Legal notice

Legal notice:

Copyright (c) Pielaszek Research, all rights reserved.
The above materials, including auxiliary resources, are subject to Publisher's copyright and the Author(s) intellectual rights. Without limiting Author(s) rights under respective Copyright Transfer Agreement, no part of the above documents may be reproduced without the express written permission of Pielaszek Research, the Publisher. Express permission from the Author(s) is required to use the above materials for academic purposes, such as lectures or scientific presentations.
In every case, proper references including Author(s) name(s) and URL of this webpage: https://science24.com/paper/29421 must be provided.

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

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

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