Current strategies for the use of affinity tags and tag removal for the purification of recombinant proteins

Arnau, José; Lauritzen, Conni; Petersen, Gitte Ebert; Pedersen, John

doi:10.1016/j.pep.2005.12.002

Cited by 553 publications

(354 citation statements)

References 107 publications

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“…Nonspecific adsorption of cellular proteins to affinity resins is reported with many commonly used purification procedures. 1,3,4 We have shown that the HIS resin exhibits a low degree of nonspecific binding, circumventing the need for supplementing the binding buffer with high concentrations of salts or eluting agents, thus simplifying the purification procedure and allowing for mild column loading conditions. This is exemplified by comparing the purification of AzHm14 using the HIS column with that of Az-Hm20 by the IMAC method.…”

Section: Discussionmentioning

confidence: 99%

“…1,2 Fusion tags exist for a variety of applications, including affinity purification, solubility enhancement, and protein detection. [1][2][3][4][5] Affinity purification methods are popular because they can dramatically reduce purification time, often require few purification steps, and can be used to obtain greater than 90% purity with high yields. 1,3,5 However, affinity-tagged proteins are purified using conditions (buffer, additives, etc.)…”

Section: Introductionmentioning

confidence: 99%

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A heme fusion tag for protein affinity purification and quantification

Asher

Bren

2010

Protein Science

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We report a novel affinity-based purification method for proteins expressed in Escherichia coli that uses the coordination of a heme tag to an L-histidine-immobilized sepharose (HIS) resin. This approach provides an affinity purification tag visible to the eye, facilitating tracking of the protein. We show that azurin and maltose binding protein are readily purified from cell lysate using the heme tag and HIS resin. Mild conditions are used; heme-tagged proteins are bound to the HIS resin in phosphate buffer, pH 7.0, and eluted by adding 200-500 mM imidazole or binding buffer at pH 5 or 8. The HIS resin exhibits a low level of nonspecific binding of untagged cellular proteins for the systems studied here. An additional advantage of the heme tag-HIS method for purification is that the heme tag can be used for protein quantification by using the pyridine hemochrome absorbance method for heme concentration determination.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A heme fusion tag for protein affinity purification and quantification

Asher

Bren

2010

Protein Science

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“…Complex aggregate formation and misfolding is to be avoided in the isolation and purification procedures. Solubilized rhIFN-γ has been purified using a variety of chromatographic and affinity techniques, including size exclusion gelfiltration [25], CM-Sepharose [27], MonoS [28], S-Sepharose [29], and cation exchange [30] chromatography, sometimes incorporating monoclonal antibodies [31], and affinity tags [32].…”

Section: Introductionmentioning

confidence: 99%

Increased yield of high purity recombinant human interferon-γ utilizing reversed phase column chromatography

Reddy

Narala

et al. 2007

Protein Expression and Purification

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Increasing therapeutic applications for recombinant human interferon-γ (rhIFN-γ), an antiviral proinflammatory cytokine, has broadened interest in optimizing methods for its production and purification. We describe a reversed phase chromatography (RPC) procedure using Source-30 ™ matrix in the purification of rhIFN-γ from Escherichia coli that results in a higher yield than previously reported. The purified rhIFN-γ monomer from the RPC column is refolded in Tris buffer. Optimal refolding occurs at protein concentrations between 50-100 μg/ml. This method yields greater than 90% of the dimer form with a yield of 40 mg g −1 cell mass. Greater than 99% purity is achieved with further purification over a Superdex G-75 column to obtain specific activities of from 2 to 4 × 10 7 IU/mg protein as determined via cytopathic antiviral assay. The improved yield of rhIFN-γ in a simple chromatographic purification procedure promises to enhance the development and therapeutic application of this biologically potent molecule.

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“…The use of tags can improve the biochemical properties and increase the yield of the produced protein, simplify renaturation conditions, increase the solubility of the produced protein, or prevent its proteolysis. However, the use of affinity tags may also have adverse impacts on labelled proteins since, for example, can alter the conformation of the labelled protein, inhibit enzyme activity, (Arnau 2006b). Therefore, when choosing an affinity tag, several inevitable points have to be considered to achieve the desired biochemical features of the protein of interest.…”

Section: Protein and Peptide Tagsmentioning

confidence: 99%

How to approach heterogeneous protein expression for biotechnological use: An overview

Jamrichová¹,

Tišáková²,

Jarábková³

et al. 2017

Nova Biotechnologica Et Chimica

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Production of recombinant proteins in Escherichia coli expression systems has shown many advantages, as well as disadvantages, especially for biotechnological and other down-stream applications. The choice of an appropriate vector depends on the gene, to be cloned for purification procedures and other analyses. Selection of a suitable production strain plays an important role in the preparation of recombinant proteins. The main criteria for the selection of the host organism are the properties of the recombinant produced protein, its subsequent use and the total amount desired. The most common problems in eukaryotic gene expression and recombinant proteins purification are, for instance, post-translational modifications, formation of disulphide bonds, or inclusion bodies. Obtaining a purified protein is a key step enabling further characterization of its role in the biological system. Moreover, methods of protein purification have been developed in parallel with the discovery of proteins and the need for their studies and applications. After protein purification, and also between the individual purification steps, it is necessary to test protein stability under different conditions over time. Shortly, all the essential points have been briefly discussed, which could be encountered during production and purification of a recombinant protein of interest, especially from eukaryotic source and expressed heterogeneously in prokaryotic production system.

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Current strategies for the use of affinity tags and tag removal for the purification of recombinant proteins

Cited by 553 publications

References 107 publications

A heme fusion tag for protein affinity purification and quantification

A heme fusion tag for protein affinity purification and quantification

Increased yield of high purity recombinant human interferon-γ utilizing reversed phase column chromatography

How to approach heterogeneous protein expression for biotechnological use: An overview

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