Several Affinity Tags Commonly Used in Chromatographic Purification

Zhao, Xinyu; Li, Guoshun; Liang, Shufang

doi:10.1155/2013/581093

Cited by 166 publications

(113 citation statements)

References 78 publications

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“…Native elution is most commonly achieved by competitive displacement of the tagged protein complex or proteolytic cleavage of the tag, releasing the complex from the affinity medium. 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 .…”

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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Section: Discussionmentioning

confidence: 99%

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

LaCava

Jiang

Rout

2016

JoVE

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“…One widely used method entails creating hybrid proteins, in which one of the protein partners plays the role of a tag (Zhao et al 2013) The use of elastin-like polypeptides seems to be an alternative to the traditional methods of protein purification. Comparison of ELPs and other affinity tags is shown in Table 2.…”

Section: For Protein Purificationmentioning

confidence: 99%

Elastin-like polypeptides as a promising family of genetically-engineered protein based polymers

Kowalczyk

Hnatuszko-Konka

Gerszberg

et al. 2014

World J Microbiol Biotechnol

109

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Elastin-like polypeptides (ELP) are artificial, genetically encodable biopolymers, belonging to elastomeric proteins, which are widespread in a wide range of living organisms. They are composed of a repeating pentapeptide sequence Val–Pro–Gly–Xaa–Gly, where the guest residue (Xaa) can be any naturally occurring amino acid except proline. These polymers undergo reversible phase transition that can be triggered by various environmental stimuli, such as temperature, pH or ionic strength. This behavior depends greatly on the molecular weight, concentration of ELP in the solution and composition of the amino acids constituting ELPs. At a temperature below the inverse transition temperature (Tt), ELPs are soluble, but insoluble when the temperature exceeds Tt. Furthermore, this feature is retained even when ELP is fused to the protein of interest. These unique properties make ELP very useful for a wide variety of biomedical applications (e.g. protein purification, drug delivery etc.) and it can be expected that smart biopolymers will play a significant role in the development of most new materials and technologies. Here we present the structure and properties of thermally responsive elastin-like polypeptides with a particular emphasis on biomedical and biotechnological application.

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“…Some short affinity tags (His tag, Stripe tag, Argo tag, and FLAG tag) have been successfully used in bacterial and yeast biosynthesis and purification systems (Agarwal et al, 2014;Zhao, Li, & Liang, 2013). However, the small tags in plant molecular pharming are characterized by their ineffectiveness, lack of scalability and high cost (Joensuu et al, 2010;Waugh, 2005).…”

Section: Fusion Tags In Plant Molecular Pharmingmentioning

confidence: 99%

Untitled

2014

Plant Sci. Today

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Plant molecular pharming is a promising concept based on the large-scale production of recombinant proteins encompassing antibodies, vaccines, enzymes and peptides for human or veterinary uses and treatments. This new branch of the biopharmaceutical industry offers practical and safety advantages over other traditional production systems. In higher plants, the complex cellular machinery makes possible synthesis and posttranslational modifications of heterologous protein macromolecules. The limiting obstacle to using this system at an industrial scale is most often the low yield of the recombinant proteins produced in host plants. To improve production levels, many studies have been focusing on the choice of plant species, tissues, organs and cell suspension cultures and/or various upstream and downstream constituents in the expression cassette. New engineering technologies in plant molecular pharming have emerged that rely on the usefulness of using soybean agglutinin (SBA), hydrophobin, zein and elastin-like peptide tags which are employed to extract and purify recombinant proteins in

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Several Affinity Tags Commonly Used in Chromatographic Purification

Cited by 166 publications

References 78 publications

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

Elastin-like polypeptides as a promising family of genetically-engineered protein based polymers

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