In selecting a method to produce a recombinant protein, a researcher is faced with a bewildering array of choices as to where to start. To facilitate decision-making, we describe a consensus 'what to try first' strategy based on our collective analysis of the expression and purification of over 10,000 different proteins. This review presents methods that could be applied at the outset of any project, a prioritized list of alternate strategies and a list of pitfalls that trip many new investigators.

Heme-degrading enzymes are involved in human diseases ranging from stroke, cancer, and multiple sclerosis to infectious diseases such as malaria, diphtheria, and meningitis. All mammalian and microbial enzymes identified to date are members of the heme oxygenase superfamily and assume similar monomeric structures with an all ␣-helical fold. Here we describe the crystal structures of IsdG and IsdI, two heme-degrading enzymes from Staphylococcus aureus. The structures of both enzymes resemble the ferredoxin-like fold and form a ␤-barrel at the dimer interface. Two large pockets found on the outside of the barrel contain the putative active sites. Sequence homologs of IsdG and IsdI were identified in multiple Gram-positive pathogens. Substitution of conserved IsdG amino acid residues either reduced or abolished heme degradation, suggesting a common catalytic mechanism. This mechanism of IsdG-mediated heme degradation may be similar to that of the structurally related monooxygenases, enzymes involved in the synthesis of antibiotics in Streptomyces. Our results imply the evolutionary adaptation of microbial enzymes to unique environments.Staphylococcus aureus acquires iron, an essential nutrient required for infection, by binding host heme-carrying proteins and extracting and transporting heme across the bacterial cell wall and plasma membrane envelope (1-3). Once inside the staphylococcal cytoplasm, heme is either incorporated into bacterial heme proteins or degraded to release iron for subsequent incorporation into polypeptides and cofactors (4 -6). One heme acquisition system, encoded by the isd (iron-regulated surface determinants) gene cluster, encodes the heme-degrading enzyme IsdG (1, 5). IsdI, a homolog of IsdG, is encoded elsewhere on the staphylococcal chromosome (5). IsdG and IsdI show no significant sequence similarity to known heme oxygenases (7-10) and do not contain the conserved N-terminal histidine or the GXXXG motif characteristic for these enzymes. Nonetheless, purified IsdG and IsdI cleave heme tetrapyrrole in the presence of suitable electron donors (2, 5). This activity can functionally substitute for the classical heme oxygenase activity and permits growth of Corynebacterium ulcerans (5) lacking the HmuO heme oxygenase on media with hemin as a sole source of iron (8). Here we address the question of whether IsdG and IsdI have structures and catalytic mechanisms similar to those of members of the heme oxygenase superfamily (11), enzymes degrading heme in the cytoplasm of eukaryotic cells and in some bacterial species such as Corynebacterium diphtheriae, Pseudomonas aeruginosa, and Neisseria spp (9). EXPERIMENTAL PROCEDURESPreparation of Proteins for Crystallization-The cells were grown at 37°C in Luria-Bertani broth in the presence of 100 g/ml ampicillin and 30 g/ml kanamycin, respectively. Expression of His-tagged fusion proteins in Escherichia coli strain BL21(DE3) (12) was induced with 1 mM D-isopropyl-␤-thiogalactoside when the optical density at 600 nm reached ϳ0.6 and incubated at 20°C ove...

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), we have developed semi-automated 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: (i) 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; [1] the second step is IMAC and buffer exchange (IMAC-II) to remove the cleaved tag and tagged TEV protease. These protocols have been implemented on multidimensional chromatography workstations and, as we have shown, many proteins can be successfully produced in large-scale. All methods and protocols used for purification, some developed by MCSG, 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 in this chapter.

A critical issue in structural genomics, and in structural biology in general, is the availability of highquality samples. The additional challenge in structural genomics is the need to produce high numbers of proteins with low sequence similarities and poorly characterized or unknown properties. 'Structural-biology-grade' proteins must be generated in a quantity and quality suitable for structure determination experiments using X-ray crystallography or nuclear magnetic resonance (NMR). The choice of protein purification and handling procedures plays a critical role in obtaining high-quality protein samples. The purification procedure must yield a homogeneous protein and must be highly reproducible in order to supply milligram quantities of protein and/or its derivative containing marker atom(s). At the Midwest Center for Structural Genomics we have developed protocols for highthroughput protein purification. These protocols have been implemented on AKTA EXPLORER 3D and AKTA FPLC 3D workstations capable of performing multidimensional chromatography. The automated chromatography has been successfully applied to many soluble proteins of microbial origin. Various MCSG purification strategies, their implementation, and their success rates are discussed in this paper.