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.

SummaryFifteen related ligation-independent cloning vectors were constructed for high-throughput cloning and purification of proteins. The vectors encode a TEV protease site for removal of tags that facilitate protein purification (his-tag) or improve solubility (MBP, GST). Specialized vectors allow coexpression and copurification of interacting proteins, or in vivo removal of MBP by TVMV protease to improve screening and purification. All target genes and vectors are processed by the same protocols, which we describe here.

Production of milligram quantities of numerous proteins for structural and functional studies requires an efficient purification pipeline. We found that the dual tag, his 6 -tag-maltose-binding protein (MBP), intended to facilitate purification and enhance proteins' solubility, disrupted such a pipeline, requiring additional screening and purification steps. Not all proteins rendered soluble by fusion to MBP remained soluble after its proteolytic removal, and in those cases where the protein remained soluble, standard purification protocols failed to remove completely the stoichiometric amount of his 6 -tagged MBP generated by proteolysis. Both liabilities were alleviated by construction of a vector that produces fusion proteins in which MBP, the his 6 -tag and the target protein are separated by highly specific protease cleavage sites in the configuration MBP-site-his 6 -site-protein. In vivo cleavage at the first site by co-expressed protease generated untagged MBP and his 6 -tagged target protein.Proteins not truly rendered soluble by transient association with MBP precipitated, and untagged MBP was easily separated from the his-tagged target protein by conventional protocols. The second protease cleavage site allowed removal of the his 6 -tag. KeywordsHigh-throughput; Structural genomics; Maltose-binding protein; TVMV protease; Ligationindependent cloningThe burgeoning genomic information now available makes vast numbers of proteins accessible for structural and functional studies, and many large-scale projects have developed automated protocols for amplifying, cloning, and expressing genes, and for screening proteins for desirable properties [1][2][3][4][5]. Similar strides have been made in streamlining protein purification, but production of sufficient material for detailed structural and functional characterization remains labor-intensive and time-consuming [3,4,6,7]. Typically, purification is facilitated by fusing proteins to affinity tags, most commonly a his-tag, which allows purification by immobilized metal-ion affinity chromatography (IMAC,[8]). Additional tags are often attached to improve proteins' solubility, such as maltose-binding protein (MBP) [2][3][4]9,10]. In typical protein production pipelines, the resulting fusion proteins are first screened for ☆ This manuscript has been created by the University of Chicago as operator of Argonne National Laboratory under Contract No.W-31-109-ENG-38 with the US Department of Energy. The US government retains for itself, and others acting on its behalf, a paid-up, nonexclusive, irrevocable worldwide license in said article to reproduce, prepare derivative works, distribute copies to the public, and perform publicly and display publicly, by or on behalf of the government. The US Government's right to retain a nonexclusive royaltyfree license in and to the copyright covering this paper, for governmental purposes, is acknowledged. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript solubility, then purified by semi-robotic p...

Clones for human prothymosin a have been identified in cDNA libraries from staphylococcal enterotoxin A-stimulated normal human lymphocytes and from simian virus 40-transformed fibroblasts. The 1198-base-pair fibroblast clone has been sequenced. The encoded protein is highly acidic (54 residues out of 111) and shares >90% sequence homology with rat prothymosin a. The peptide "hormone" thymosin al appears at positions 2-29 of the prothymosin a amino acid sequence. There is no N-terminal signal peptide.

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.

Background: asbABCDEF mediates petrobactin production and facilitates anthrax virulence. Results: Purified AsbA-E proteins reconstituted petrobactin assembly in vitro. The crystal structure and enzymatic studies of AsbB highlight its function and role in the siderophore pathway. Conclusion: AsbB characterization demonstrated reaction flexibility and substrate positions in the binding pocket. Significance: Siderophore synthetases represent promising antimicrobial targets, and characterization of these versatile enzymes enables creation of novel compounds.

Candida tropicalis ATCC 20336 can grow on fatty acids or alkanes as its sole source of carbon and energy, but strains blocked in ␤-oxidation convert these substrates to long-chain ␣,-dicarboxylic acids (diacids), compounds of potential commercial value (Picataggio et al., Biotechnology 10:894-898, 1992). The initial step in the formation of these diacids, which is thought to be rate limiting, is -hydroxylation by a cytochrome P450 (CYP) monooxygenase. C. tropicalis ATCC 20336 contains a family of CYP genes, and when ATCC 20336 or its derivatives are exposed to oleic acid (C 18:1 ), two cytochrome P450s, CYP52A13 and CYP52A17, are consistently strongly induced (Craft et al., this issue). To determine the relative activity of each of these enzymes and their contribution to diacid formation, both cytochrome P450s were expressed separately in insect cells in conjunction with the C. tropicalis cytochrome P450 reductase (NCP). Microsomes prepared from these cells were analyzed for their ability to oxidize fatty acids. CYP52A13 preferentially oxidized oleic acid and other unsaturated acids to -hydroxy acids. CYP52A17 also oxidized oleic acid efficiently but converted shorter, saturated fatty acids such as myristic acid (C 14:0 ) much more effectively. Both enzymes, in particular CYP52A17, also oxidized -hydroxy fatty acids, ultimately generating the ␣,-diacid. Consideration of these different specificities and selectivities will help determine which enzymes to amplify in strains blocked for ␤-oxidation to enhance the production of dicarboxylic acids. The activity spectrum also identified other potential oxidation targets for commercial development.

Correspondence to: Andrzej Joachimiak, andrzejj@anl.gov. The submitted manuscript has been created by the University of Chicago as operator of Argonne National Laboratory under Contract No. W-31-109-ENG-38 with the U.S. Department of Energy. The U.S. government retains for itself, and others acting on its behalf, a paid-up, nonexclusive, irrevocable worldwide license in said article to reproduce, prepare derivative works, distribute copies to the public, and perform publicly and display publicly, by or on behalf of the government. High-throughput structural genomics projects seek to delineate protein structure space by determining the structure of representatives of all major protein families. Generally this is accomplished by processing numerous proteins through standardized protocols, for the most part involving purification of N-terminally His-tagged proteins. Often proteins that fail this approach are abandoned, but in many cases further effort is warranted because of a protein's intrinsic value. In addition, failure often occurs relatively far into the path to structure determination, and many failed proteins passed the first critical step, expression as a soluble protein. Salvage pathways seek to recoup the investment in this subset of failed proteins through alternative cloning, nested truncations, chemical modification, mutagenesis, screening buffers, ligands and modifying processing steps. To this end we have developed a series of ligation-independent cloning expression vectors that append various cleavable C-terminal tags instead of the conventional N-terminal tags. In an initial set of 16 proteins that failed with an N-terminal appendage, structures were obtained for C-terminally tagged derivatives of five proteins, including an example for which several alternative salvaging steps had failed. The new vectors allow appending C-terminal His 6 -tag and His 6 -and MBP-tags, and are cleavable with TEV or with both TEV and TVMV proteases. NIH Public Access