Abstract
Protein crystallization and subsequent X-ray diffraction analysis of the three-dimensional structure are necessary for elucidation of the biological functions of proteins and effective rational drug design. Therefore, controlling protein crystallization is important to obtain high resolution X-ray diffraction data. Here, a simple microfluidic method using chips with 10 and 50 μm high crystallization chambers to selectively form suitable single protein crystals for X-ray analysis is demonstrated. As proof of concept, three different types of proteins: lysozyme, glucokinase from Pseudoalteromonas sp. AS-131 (PsGK), and NADPH-cytochrome P450 oxidoreductase–heme oxygenase complex were crystallized. We demonstrate that the crystal growth orientation depends on the height of the crystallization chamber regardless of the protein type. Our results suggest that the confined micro space induces the protein molecules to adhere to a specific crystal face and affects the growth kinetics of each crystal face. The present microfluidic-based protein crystallization method can reform a suitable single protein crystal for X-ray analysis from aggregates of needle-shaped protein crystals.
Highlights
In the last decade, a variety of techniques and methods have been developed to facilitate protein crystallization experiments.[3,4,5] Microfluidics-based platforms offer promising approaches for the screening of protein crystallization conditions and in situ X-ray analysis.[6,7,8,9,10,11] A microfluidic chip is able to reduce the consumption of protein samples and crystallization reagents because the required volume of one protein crystallization trial is a few nanoliters to a few tens of nanoliters
We demonstrate that the crystal growth orientation depends on the height of the crystallization chamber regardless of the protein type
Microfluidic chips were fabricated by standard soft lithography with minor modifications.[20]
Summary
A variety of techniques and methods have been developed to facilitate protein crystallization experiments.[3,4,5] Microfluidics-based platforms offer promising approaches for the screening of protein crystallization conditions and in situ X-ray analysis.[6,7,8,9,10,11] A microfluidic chip is able to reduce the consumption of protein samples and crystallization reagents because the required volume of one protein crystallization trial is a few nanoliters to a few tens of nanoliters. A simple microfluidic method using chips with 10 and 50 μm high crystallization chambers to selectively form suitable single protein crystals for X-ray analysis is demonstrated. We report a simple method using microfluidic chips with 10–50 μm deep crystallization chambers to form suitable protein crystals for X-ray protein crystallography.
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