Approach for growth of high-quality and large protein crystals

Hiroyoshi Matsumura,Yusuke Mori,Ryota Murai,Keisuke Kakinouchi,Tsuyoshi Inoue,Mika Hirose,Mihoko Maruyama,Shigeru Sugiyama,Kazufumi Takano,Hiroaki Adachi,Satoshi Murakami

doi:10.1107/s090904951003445x

Hiroyoshi Matsumura, Yusuke Mori + Show 9 more

Open Access

https://doi.org/10.1107/s090904951003445x

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Abstract

Three crystallization methods for growing large high-quality protein crystals, i.e. crystallization in the presence of a semi-solid agarose gel, top-seeded solution growth (TSSG) and a large-scale hanging-drop method, have previously been presented. In this study the effectiveness of crystallization in the presence of a semi-solid agarose gel has been further evaluated by crystallizing additional proteins in the presence of 2.0% (w/v) agarose gel, resulting in complete gelification with high mechanical strength. In TSSG the seed crystals are hung by a seed holder protruding from the top of the growth vessel to prevent polycrystallization. In the large-scale hanging-drop method, a cut pipette tip was used to maintain large-scale droplets consisting of protein-precipitant solution. Here a novel crystallization method that combines TSSG and the large-scale hanging-drop method is reported. A large and single crystal of lysozyme was obtained by this method.

Highlights

The three-dimensional structures of proteins are widely utilized to interpret the biological phenomena involved in proteins and to develop new pharmaceutical products, since the structures demonstrate how the proteins function and how they interact with specific molecules
The production of large high-quality protein crystals remains a bottleneck in X-ray and neutron crystallography
Crystallographers have developed many protein crystallization methods, such as microbatch, dialysis, counterdiffusion and hanging-drop, as well as sitting-drop vapor diffusion methods (Chayen, 1996, 1999; D’Arcy et al, 1996; McPherson, 1982, 1999). Each of these current techniques has its merits, limitations still remain. To overcome these limitations we previously presented novel crystallization methods, including crystallization in the presence of the semisolid agarose gel (Hasenaka et al, 2009; Sugiyama et al, 2009a,b; Tanabe et al, 2009), top-seeded solution growth (TSSG) (Shimizu et al, 2009, 2010) and a large-scale hangingdrop method (Kakinouchi et al, 2010), to grow large highquality protein crystals

Summary

Introduction

Neutron protein crystallography is capable of visualizing hydrogen atoms and hydration water molecules to yield a more precise understanding of the protein structure. Very large crystals (e.g. 1–10 mm3) (Myles, 2006) of the target proteins are required to determine the structures by neutron crystallography. To prevent polycrystallization during growth of large protein crystals we previously applied TSSG for protein crystallization. TSSG effectively prevents polycrystallization to obtain a single large protein crystal, this method requires a large amount of protein and precipitating agents (e.g. 1 ml). The large-scale hanging-drop method was developed to resolve these issues (Kakinouchi et al, 2010). This simple method can be applied to the vapor diffusion method, permitting the concentration of the precipitant agent to be adjusted by vapor diffusion. We combined the large-scale hangingdrop method with TSSG to overcome the limitations

Combination of the large-scale hanging-drop method and TSSG

Findings

18 Hiroyoshi Matsumura et al Growth of large protein crystals