Laboratory Automation and High- Throughput Chemistry High-Throughput Protein Purification and Quality Assessment for Crystallization

The ultimate goal of structural biology is to understand the structural basis of proteins in cellular processes. In structural biology, the most critical issue is the availability of high-quality samples. Structural biology-grade proteins must be generated in the quantity and quality suitable for structure determination using x-ray crystallography or nuclear magnetic resonance (NMR) spectroscopy. The purification procedures must reproducibly yield homogeneous proteins or their derivatives containing marker atom(s) in milligram quantities. The choice of protein purification and handling procedures plays a critical role in obtaining high-quality protein samples. With structural genomics emphasizing a genome-based approach in understanding protein structure and function, a number of unique structures covering most of the protein folding space have been determined, and new technologies with high efficiency have been developed. At the Midwest Center for Structural Genomics (MCSG) of Argonne National Laboratory, the authors, Kim et al., have developed semiautomated protocols for high-throughput parallel protein expression and purification. A protein, expressed as a fusion with a cleavable affinity tag, is purified in two consecutive immobilized metal affinity chromatography (IMAC) steps. The first step is an IMAC coupled with buffer exchange, or size exclusion chromatography (IMAC-I), followed by the cleavage of the affinity tag using the highly specific Tobacco Etch Virus (TEV) protease. The second step is IMAC and buffer exchange (IMAC-II) to remove the cleaved tag and tagged TEV protease. These protocols are implemented on multidimensional chromatography workstations and show that many proteins can be successfully produced in large scale. All methods and protocols used for purification, some developed by MCSG and others adopted and integrated into the MCSG purification pipeline and more recently the Center for Structural Genomics of Infectious Diseases (CSGID) purification pipeline, are discussed. (Kim, Y., et al., Methods 2011, 55, 12–28) Comparison of Commercial Extraction Systems and PCR Assays for Quantification of Epstein-Barr Virus DNA Load in Whole Blood