by using high-viscous reagent such as polyethylene glycol (PEG) 8000 for crystallization solution,
and 'β' can be increased by using highly purified protein sample for crystallization. Researchers
now become able to estimate microgravity effects and optimize the crystallization condition
prior to performing microgravity experiments by referring to this β/D value.
Here are some results of the successful crystallization experiments in space.
The crystals of Aspergillus oryzae alpha-amylase were obtained as
cluster-like crystals that diffracted up to 1.4 Å resolution on the
ground so far. However, after the further purification of the
protein sample using FPLC and changing the precipitant from salt
to high viscous polyethylene glycol (PEG) 8000, high-quality
crystals were obtained. These crystals diffracted up to 0.79 Å
resolution by visual inspection; and a full X-ray diffraction dataset
could be obtained up to 0.92 Å resolution. After the data analysis,
the electron density corresponding to hydrogen atoms were
visualized.
Hematopoietic prostaglandin D synthase (H-PGDS) and Lipocalin-
type prostaglandin D synthase (L-PGDS) are both clinically important drug target proteins
provided by Professor Urade of University of Tsukuba.
H-PGDS was crystallized in space 12 times with more than 20 inhibitors since 1997. It was
difficult to obtain good crystals of H-PGDS initially. Accordingly, scientists used the same
strategy, which applied to the crystallization of alpha-amylase, to the crystallization of H-PGDS
(using PEG as a high-viscous precipitant and highly purified protein sample). Afterward,
investigators were able to obtain high-quality crystals of H-PGDS with novel inhibitors that
diffracted X-ray waves up to 1.1 Å. Those novel inhibitors are expected to be candidates for
novel drug designs.
L- PGDS with a C65A mutation was previously crystallized with citrate or malonate as a
precipitant, and the X-ray crystal structure was determined at 2.0 Å resolution. Then, scientists
attempted to obtain high-quality crystals of the C64A mutant under microgravity environment
by using the same conditions as used in the previous study, but they could not obtain a
satisfactory results. Therefore the same strategy as mentioned above was used, and then high-
quality crystals could be obtained in microgravity, which diffracted at around 1.0 Å resolution.
The crystal quality was markedly improved through the use of a high-viscosity precipitant
solution in microgravity, in combination with the use of a highly purified protein.
These examples are a part of JAXA’s procedure for growing high-quality protein crystals. From
the purification of a protein sample to the high-resolution X-ray data collection, including the
optimization of crystallization condition on the ground, JAXA has established a sequence of
experimental steps for successful crystallization in space.
Electron density map of alpha-
amylase Image courtesy of
Professor Nakagawa, Osaka
University.