Protein crystal growth with stirring solution

Hiroaki Adachi,^a Kazufumi Takano,^b Masashi Yoshimura,^a Yusuke Mori,^a and Takatomo Sasaki^a

^aDepartment of Electrical Engineering and Venture Business Laboratory, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan; ^bDepartment of Material and Life Science, Osaka University, and PRESTO, JST, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan (adachi@ssk.pwr.eng.osaka-u.ac.jp)

The production of protein crystals with suitable diffraction quality and size is a rate-limiting step for determining the three-dimensional structure of proteins at atomic resolution. We propose a protein crystal growth with stirring the solution, which is entirely different approach compared with the conventional method, leading to an effective growth of protein crystals. Crystallographers believe that protein crystal growth must be under a still condition, and it is ultimately performed under a microgravity condition due to reducing convective flow. It is important that the protein solution must be stirred gently in protein crystal growth because directly stirring a protein solution causes problems such as spontaneous nucleation, protein denaturation and damaged crystals. We developed two methods for growing large, high-quality protein crystals. One is FAST (floating and stirring technique) using the Two-Liquid System[1] and a magnetic stirrer. The other is Micro-Stirring technique using a rotary shaker.

FAST realizes mild stirring suitable for protein crystal growth by stirring the liquid of the lower layer, which is primarily an indirect stirring method. The stirring intensity is changed to optimize the growth conditions for a specific rotation rate, shape of stirring bar, and volume of lower liquid. Using this method together with slow cooling, we obtained a 3.0 mm-long lysozyme crystal in 20 days from a seed crystal[2]. This is larger than the 2.0 mm-long crystal grown without stirring. When the protein solution is kept still, protein concentration gradually increases in the lower part of the solution. Consequently, excess protein solute that is not supplied to the seed crystal leads to additional spontaneous nucleation. Stirring the protein solution accelerates the growth of the protein crystal and prevents additional spontaneous nucleation.

On the other hand, Micro-Stirring enables to stir multiple micro-scale samples in the growth of protein crystals by the sitting-drop and floating-drop[3] vapor diffusion techniques. This is also effective in reducing the number of crystals and in growing large crystals compared to the conventional vapor diffusion technique[4]. We also expect that stirring the solution will improve crystallinity.

References

1 Adachi, H., Watanabe, T., Yoshimura, M., Mori, Y. and Sasaki, T. (2002) Jpn. J. Appl. Phys. 41, L726-L728.

2 Adachi, H., Takano, K., Yoshimura, M., Mori, Y. and Sasaki, T. (2002) Jpn. J. Appl. Phys. 41, L1025-L1027.

3 Adachi, H., Takano, K., Morikawa, M., Kanaya, S., Yoshimura, M., Mori, Y. and Sasaki, T. (2003) Acta. Cryst. D59, 194-196.

4 Adachi, H., Takano, K., Yoshimura, M., Mori, Y. and Sasaki, T. (2003) Jpn. J. Appl. Phys. 42, L314-L315.