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.

Fig. 1

SDS-PAGE of IDP01350 (MW: 23.6 kDa, lanes 1–3, 5, and 6) and IDP01460 (44.6 kDa, lanes 7–11) purified by the process described: lanes 1 and 7—crude extract, lanes 2 and 8—after IMAC-I, lanes 3 and 9—after TEV cleavage, lanes 5 and 10—after IMAC-II, lanes 6 and 11—elution with 250 mM imidazole after IMAC-II, and lane 4—molecular weight markers (EZ-Run Fisher), from the lower to higher one (marked red triangle) 10, 15, 20, 25, 30, 40, 50, 60, 70 kDa. For IDP1350, TEV cleavage was not complete, but after IMAC-II, the cleaved IDP1350 was cleaned well.

Fig. 2

(A) Size exclusion profile of IDP indicating a small organic molecule eluted near the end of the column volume. (B) Before size exclusion chromatography, the small organic molecule is shown by high absorbance of OD₂₆₀. (C) After size exclusion chromatography, the species absorbing at 260 nm is removed.

Fig. 3

Gadgets used in the on-column cleavage: (A) AKTAxpress with a 26/60 size exclusion column, (B) a 5 ml HisTrap Ni-column wrapped a heat strip, and (C) temperature controller.

Fig. 4

SDS-PAGE showing GroEL, indicated in red arrow near 57 kDa, coeluted with target proteins in the IMAC-I step. Molecular weight marker indicates (in red triangles) 5, 10, 15, 25, 35, 50, 70, 90 kDa.