Automation of protein purification for structural genomics

Kim, Young Chang; Dementieva, I.; Zhou, Min; Wu, R.; Lezondra, L.; Quartey, P.; Joachimiak, G.; Korolev, O.; Li, Hui; Joachimiak, Andrzej

doi:10.1023/b:jsfg.0000029206.07778.fc

Cited by 103 publications

(108 citation statements)

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“…Cultures were incubated overnight, harvested the next morning, suspended in lysis buffer (50 mM Hepes, pH 7.8, containing 500 mM NaCl, 10 mM imidazole, 10 mM β-mercaptoethanol, and 5% glycerol), and lysed by sonication. Proteins were purified by established protocols [6]. …”

Section: Production Of Selenomethionyl Proteins In Non-sterile Enrichmentioning

confidence: 99%

“…4 shows the partial purification of three proteins through the first IMAC step and is representative of the strong expression, efficient cleavage by co-expressed TVMV protease, and complete removal of MBP typically obtained. The three proteins were further purified by subtractive IMAC to remove trace Escherichia coli proteins [6], and analyzed for selenomethionine incorporation. Amino acid analysis failed to detect methionine in any of the three proteins, consistent with selenomethionine incorporation of 90% or more [21].…”

Section: Production Of Selenomethionyl Proteins From Pmcsg19mentioning

confidence: 99%

“…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]).…”

Section: Introductionmentioning

confidence: 99%

“…A second step, such as subtractive IMAC, then removes contaminating host proteins. When standard protocols of this design, as implemented by the Midwest Center for Structural Genomics (MCSG) [6], were applied to targets appended with N-terminal his 6 -MBP tags, complications arose because of false positives (proteins scored as soluble in screens of fusion proteins but insoluble after removal of MBP) and by failure of the secondary IMAC step to remove completely the his 6 -MBP generated by TEV cleavage. Here we describe a new vector that alleviates these problems without modification of established screening and purification protocols.…”

mentioning

confidence: 99%

“…solubility, then purified by semi-robotic protocols in which the tags are removed by a specific protease such as the tobacco etch virus (TEV) protease [4,6,11,12]. A second step, such as subtractive IMAC, then removes contaminating host proteins.…”

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confidence: 99%

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An expression vector tailored for large-scale, high-throughput purification of recombinant proteins

Donnelly

Zhou

Millard

et al. 2006

Protein Expression and Purification

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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...

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Section: Production Of Selenomethionyl Proteins In Non-sterile Enrichmentioning

confidence: 99%

Section: Production Of Selenomethionyl Proteins From Pmcsg19mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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An expression vector tailored for large-scale, high-throughput purification of recombinant proteins

Donnelly

Zhou

Millard

et al. 2006

Protein Expression and Purification

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159

172

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Structural genomics – expanding protein structural universe

Joachimiak

2005

Encyclopedia of Genetics, Genomics, Proteomics and Bioinformatics

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Structural Genomics has emerged as a multidisciplinary and international effort to expand the universe of protein folds by rapidly determining novel structures. This initiative takes advantage of the genome sequencing projects and technological advances in genomic analysis, molecular biology, proteomics, and structural biology. The international Structural Genomics efforts were initiated, in part, to achieve a more rapid understanding of sequence/structure relationships with the ultimate goal to make it feasible to predict an accurate 3D protein structure on the basis of sequence alone, and to elucidate novel biological function directly from primary genomic sequence. A number of Structural Genomics centers have been established to test and improve existing and develop new technologies. These centers developed the protein structure determination pipelines that include target selection, gene cloning and expression, protein and crystal production, structure determination, and structural and functional analysis. These pipelines are capable of determining hundreds of novel protein structures. Structural Genomics is rapidly expanding the universe of protein folds, creating new reagents, comprehensive libraries of expression clones, and databases for broad use. New high‐throughput technologies supported by robotics and automation in molecular biology, protein expression, purification, crystallization, and structure determination are being disseminated to the public and, therefore, contribute to the acceleration of new discoveries in biology. Ultimately, Structural Genomics will become a major component of and contributor to the next structural biology challenge – visualization of all major subcellular macromolecular assemblies of the cell.

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Affinity Fusions: Gene Expression

Gräslund

Hammarstrom

2009

Encyclopedia of Industrial Biotechnology

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In response to the rapidly growing field of proteomics, the use of recombinant proteins has increased greatly in recent years. Since proteins have very diverse physiological properties, they are not naturally adjusted to generic methods. Affinity tags, providing a common handle for all kinds of proteins, have consequently become indispensable tools for structural and functional analysis of whole proteomes. Although originally developed to facilitate the detection and purification of recombinant proteins, it has become clear in recent years that affinity tags can also have positive impact on the yield, solubility, in vivo half‐life, and even the folding of their fusion partners. However, no single affinity tag is optimal with respect to all these parameters. Each has its strengths and weaknesses and therefore a suitable tag must be chosen for each application. An alternative would be the use of combinatorial tags and thus combining several desired properties.

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Automation of protein purification for structural genomics

Cited by 103 publications

References 22 publications

An expression vector tailored for large-scale, high-throughput purification of recombinant proteins

An expression vector tailored for large-scale, high-throughput purification of recombinant proteins

Structural genomics – expanding protein structural universe

Affinity Fusions: Gene Expression

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