An overview of enzymatic reagents for the removal of affinity tags

Waugh, David S.

doi:10.1016/j.pep.2011.08.005

Cited by 303 publications

(239 citation statements)

References 146 publications

(172 reference statements)

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“…The His 6 tag was removed by cleavage with tobacco etch virus protease (expressed and purified in-house per Ref. 36) overnight at 4°C during dialysis against 0.5 M NaCl, 10 mM HEPES (pH 7.5), and 0.5 mM tris [2-carboxyethyl]phosphine. The sample was passed over nickel-nitrilotriacetic acid resin and the flow-through was collected.…”

Section: Enzyme Cloning and Expressionmentioning

confidence: 99%

Structure-Function Analysis of a Mixed-linkage β-Glucanase/Xyloglucanase from the Key Ruminal Bacteroidetes Prevotella bryantii B14

McGregor

Morar

Fenger

et al. 2016

Journal of Biological Chemistry

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The recent classification of glycoside hydrolase family 5 (GH5) members into subfamilies enhances the prediction of substrate specificity by phylogenetic analysis. However, the small number of well characterized members is a current limitation to understanding the molecular basis of the diverse specificity observed across individual GH5 subfamilies. GH5 subfamily 4 (GH5_4) is one of the largest, with known activities comprising (carboxymethyl)cellulases, mixedlinkage endo-glucanases, and endo-xyloglucanases. Through detailed structure-function analysis, we have revisited the characterization of a classic GH5_4 carboxymethylcellulase, PbGH5A (also known as Orf4, carboxymethylcellulase, and Cel5A), from the symbiotic rumen Bacteroidetes Prevotella bryantii B 1 4. We demonstrate that carboxymethylcellulose and phosphoric acid-swollen cellulose are in fact relatively poor substrates for PbGH5A, which instead exhibits clear primary specificity for the plant storage and cell wall polysaccharide, mixedlinkage ␤-glucan. Significant activity toward the plant cell wall polysaccharide xyloglucan was also observed. Determination of PbGH5A crystal structures in the apo-form and in complex with (xylo)glucan oligosaccharides and an active-site affinity label, together with detailed kinetic analysis using a variety of well defined oligosaccharide substrates, revealed the structural determinants of polysaccharide substrate specificity. In particular, this analysis highlighted the PbGH5A active-site motifs that engender predominant mixed-linkage endo-glucanase activity vis à vis predominant endo-xyloglucanases in GH5_4. However the detailed phylogenetic analysis of GH5_4 members did not delineate particular clades of enzymes sharing these sequence motifs; the phylogeny was instead dominated by bacterial taxonomy. Nonetheless, our results provide key enzyme functional and structural reference data for future bioinformatics analyses of (meta)genomes to elucidate the biology of complex gut ecosystems.

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Section: Enzyme Cloning and Expressionmentioning

confidence: 99%

Structure-Function Analysis of a Mixed-linkage β-Glucanase/Xyloglucanase from the Key Ruminal Bacteroidetes Prevotella bryantii B14

McGregor

Morar

Fenger

et al. 2016

Journal of Biological Chemistry

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“…Before a size exclusion chromatography (SEC), the tagged--OAS1 was cleaved by an in--house purified tobacco etch virus (TEV) protease. This protease targets the TEV protease recognition site (E--N--L--Y--F--Q--S), which is present preceding the OAS1 [344]. The yield for purified soluble OAS1 eluted from the SEC column per liter of LB broth is approximately 2.0 mg. Mutant versions of OAS1 (described in section 1.2.6) were also purified following exactly the same steps as the wild type OAS1.…”

Section: Expression and Purification Of Oas1 In E Colimentioning

confidence: 99%

Characterization of the termini of the West Nile virus genome and their interactions with the small isoform of the 2′ 5′-oligoadenylate synthetase family

Deo

Patel

Chojnowski

et al. 2015

Journal of Structural Biology

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confirmed the specificity of the interaction. Solution conformations of both the 5'--TR RNA of WNV and OAS1 were then elucidated using small--angle x--ray scattering. I also report that the 3' terminal region (3'--TR) is able to mediate specific interaction with and activation of OAS1. Binding and kinetic experiments identified a specific stem loop within the 3'--TR that is sufficient for activation of the enzyme. The solution confirmation of the 3'--terminal region was determined by small angle X--ray scattering, and computational models suggest a conformationally restrained structure comprised of a helix and short stem loop. Structural investigation of the 3'--ii TR in complex with OAS1 is also presented. Finally, we show that genome cyclization by base pairing between the 5'--and 3'--TRs, a required step for replication, is not sufficient to protect WNV from OAS1 recognition. The purity, monodispersity and homogeneity of all samples subjected to SAXS analysis were evaluated using dynamic light scattering and/or analytical ultra--centrifuge. These data provide a framework for understanding recognition of the highly structured terminal regions of a flaviviral genome by an innate immune enzyme.iii

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“…Several affinity systems comprised of small epitope tags exist, for which the epitope itself is available as peptide useful for competitive elution of tagged protein complexes 60 . Likewise, several proteases are available to specifically cleave cognate sites strategically placed in affinity tagged fusion proteins 61 . Depending on the specifics of the chosen affinity systems, the appropriate elution scheme may be adopted.…”

Section: Discussionmentioning

confidence: 99%

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

LaCava

Jiang

Rout

2016

JoVE

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Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this technique is also frequently referred to as immunoprecipitation. Affinity capture can be applied in a bench-scale and in a high-throughput context. When coupled with protein mass spectrometry, affinity capture has proven to be a workhorse of interactome analysis. Although there are potentially many ways to execute the numerous steps involved, the following protocols implement our favored methods. Two features are distinctive: the use of cryomilled cell powder to produce cell extracts, and antibody-coupled paramagnetic beads as the affinity medium. In many cases, we have obtained superior results to those obtained with more conventional affinity capture practices. Cryomilling avoids numerous problems associated with other forms of cell breakage. It provides efficient breakage of the material, while avoiding denaturation issues associated with heating or foaming. It retains the native protein concentration up to the point of extraction, mitigating macromolecular dissociation. It reduces the time extracted proteins spend in solution, limiting deleterious enzymatic activities, and it may reduce the non-specific adsorption of proteins by the affinity medium. Micron-scale magnetic affinity media have become more commonplace over the last several years, increasingly replacing the traditional agarose-and Sepharose-based media. Primary benefits of magnetic media include typically lower nonspecific protein adsorption; no size exclusion limit because protein complex binding occurs on the bead surface rather than within pores; and ease of manipulation and handling using magnets.

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An overview of enzymatic reagents for the removal of affinity tags

Cited by 303 publications

References 146 publications

Structure-Function Analysis of a Mixed-linkage β-Glucanase/Xyloglucanase from the Key Ruminal Bacteroidetes Prevotella bryantii B14

Structure-Function Analysis of a Mixed-linkage β-Glucanase/Xyloglucanase from the Key Ruminal Bacteroidetes Prevotella bryantii B14

Characterization of the termini of the West Nile virus genome and their interactions with the small isoform of the 2′ 5′-oligoadenylate synthetase family

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

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