Abstract
The attenuation of sedimentation and convection in microgravity can sometimes decrease irregularities formed during macromolecular crystal growth. Current terrestrial protein crystal growth (PCG) capabilities are very different than those used during the Shuttle era and that are currently on the International Space Station (ISS). The focus of this experiment was to demonstrate the use of a commercial off-the-shelf, high throughput, PCG method in microgravity. Using Protein BioSolutions’ microfluidic Plug Maker™/CrystalCard™ system, we tested the ability to grow crystals of the regulator of glucose metabolism and adipogenesis: peroxisome proliferator-activated receptor gamma (apo-hPPAR-γ LBD), as well as several PCG standards. Overall, we sent 25 CrystalCards™ to the ISS, containing ~10,000 individual microgravity PCG experiments in a 3U NanoRacks NanoLab (1U = 103 cm.). After 70 days on the ISS, our samples were returned with 16 of 25 (64%) microgravity cards having crystals, compared to 12 of 25 (48%) of the ground controls. Encouragingly, there were more apo-hPPAR-γ LBD crystals in the microgravity PCG cards than the 1g controls. These positive results hope to introduce the use of the PCG standard of low sample volume and large experimental density to the microgravity environment and provide new opportunities for macromolecular samples that may crystallize poorly in standard laboratories.
Highlights
Biochemical macromolecules are fundamental components of all living things
Filling and Freezing of Plug MakerTM CrystalCardsTM Similar to other available high-throughput fluid handling devices (e.g. TTP Labtech's Mosquito®, Art Robbins Instruments’ Gryphon, etc.) the Plug MakerTM system provided a straightforward, process of creating a large variety of conditions for protein crystal growth (PCG) using about 10 nL of protein per plug (~2-4 μL/card)
Almost all methods of μg PCG required samples to be stable in solution and was subjected to weeks of storage before loading, launch and travel to the microgravity environment
Summary
Biochemical macromolecules are fundamental components of all living things. Understanding a macromolecule’s threedimensional structure provides a deeper understanding of its function and relationship to other components that are responsible for maintaining life. Completion of the Human Genome Project in 2003 led to the formation of large structural genomic programs such as the NIH’s Protein Structure Initiative, Japan’s RIKEN and the Structural Genomics Consortium These collaborative “structure factories” have been crucial in reducing the cost of determining a macromolecular model as well as driving the production of effective technology and methodologies [3,4]. Some of their accomplishments include: more efficient cloning, expression, and purification methods; low volume, high throughput screening for solubility and crystal growth; fluid handling robots; and computational programs for collecting data and solving structures. These developments have quickly altered the landscape of pharmaceutical and academic structure laboratories, allowing for unprecedented contributions to the body of structural knowledge [5,6,7]
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