DRUG DEVELOPMENT continued a b
Figure 2 – Proteinase K crystals: a) obtained in one of the original drops (200–250 µm) and b) an optimized single crystal, well defined and larger in size (400 µm).
Unlike conditional manual optimization or optimization with a dedicated robot (e.g., the dragonfly), here the drops are equilibrating against an “alternative” reservoir, which is the original hit condition. This is because the reagents are multiaspirated from columns of a source plate and mixed directly with the protein, rather than having the components mixed in a source plate well reservoir.
Experiment 2: Optimizing crystals of a novel
proteobacteria protein In a second experiment, crystals of a novel protein found in carboxy- some-containing autotrophic proteobacteria were optimized using this method. These proteobacteria are potentially useful as bioremediation organisms (naturally occurring organisms that break down hazardous substances into less toxic or nontoxic substances). Modifying this protein could enhance productivity of the organism and optimize its suitability for contaminated waste treatment.1,2
The protein was cloned and purified in collaboration with Nicole Wheatley of UCLA. The original crystals were obtained in a hanging-drop format containing a 1:1 mixture of 25 mg/mL protein (in a buffer of 10 mM tris, pH 7.6, 50 mM NaCl) to a reservoir condition containing 4 M (NH4
)2 SO4 and
1 M bis-tris, pH 5.5 (Figure 3a). For this optimization, the concentration of the protein was kept constant, while the pH was increased on the y-axis of the optimization plate and the concentration of the AmSO4
was increased
on the x-axis. Previously, both manual and optimization under oil were set up, but only very poor crystals were obtained where the needles were too thin to even harvest.
Diffraction data were collected on some of the best single and sharp- edged optimized crystals at the UCLA X-ray crystallography core facility at a wavelength of 1.54 Å. The optimized crystals resulted in improving the resolution from 2.5 to 2.1 Å (Figure 3b). While the refinement resulted in larger crystals of 625 µm compared to 200 µm in the original hit, the best diffracting crystals were not necessarily the largest.
Conclusion The above examples demonstrate that the refinement of the crystalliza-
tion conditions is improved by automation. Although dragonfly is the instrument of choice for fast and easy high-throughput optimization, the mosquito Crystal can successfully be used for both initial screening of the crystallization conditions and also for the optimization of the conditions, required to obtain high-resolution diffracting crystals.
AMERICAN LABORATORY • 12 • MAY 2014
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