title: Chapter 3. High-throughput protein purification for x-ray crystallography and NMR.
abstract: In structural biology, the most critical issue is the availability of high-quality samples. "Structural-biology-grade" proteins must be generated in a quantity and quality suitable for structure determination using X-ray crystallography or nuclear magnetic resonance. The additional challenge for structural genomics is the need for high numbers of proteins at low cost where protein targets quite often have low sequence similarities, unknown properties and are poorly characterized. 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. Where the ultimate goal of structural biology is the same-to understand the structural basis of proteins in cellular processes, the structural genomics approach is different in that the functional aspects of individual protein or family are not ignored, however, emphasis here is on the number of unique structures, covering most of the protein folding space and developing new technologies with high efficiency. At the Midwest Center Structural Genomics (MCSG), we have developed semiautomated protocols for high-throughput parallel protein purification. In brief, a protein, expressed as a fusion with a cleavable affinity tag, is purified in two immobilized metal affinity chromatography (IMAC) steps: (i) first IMAC coupled with buffer-exchange step, and after tag cleavage using TEV protease, (ii) second IMAC and buffer exchange to clean up cleaved tags and tagged TEV protease. Size exclusion chromatography is also applied as needed. These protocols have been implemented on multidimensional chromatography workstations AKTAexplorer and AKTAxpress (GE Healthcare). All methods and protocols used for purification, some developed in MCSG, others adopted and integrated into the MCSG purification pipeline and more recently the Center for Structural Genomics of Infectious Disease (CSGID) purification pipeline, are discussed in this chapter.
authors: Midwest Center for Structural Genomics and Structural Biology Center, Biosciences Division, Argonne National Laboratory, Argonne, IL, USA.
authors: Youngchang
authors: Y
authors: Kim
authors: Lance
authors: L
authors: Bigelow
authors: Maria
authors: M
authors: Borovilos
authors: Irina
authors: I
authors: Dementieva
authors: Erika
authors: E
authors: Duggan
authors: William
authors: W
authors: Eschenfeldt
authors: Catherine
authors: C
authors: Hatzos
authors: Grazyna
authors: G
authors: Joachimiak
authors: Hui
authors: H
authors: Li
authors: Natalia
authors: N
authors: Maltseva
authors: Rory
authors: R
authors: Mulligan
authors: Pearl
authors: P
authors: Quartey
authors: Alicia
authors: A
authors: Sather
authors: Lucy
authors: L
authors: Stols
authors: Lour
authors: L
authors: Volkart
authors: Ruiying
authors: R
authors: Wu
authors: Min
authors: M
authors: Zhou
authors: Andrzej
authors: A
authors: Joachimiak
mesh_terms: D005591:Chemical Fractionation / Q000379:methods*; D018360:Crystallography, X-Ray; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification*
chemical_list: D011506:Proteins