Affinity Tags in Protein Purification and Peptide Enrichment: An Overview

Pina, Ana Sofia; Batalha, Íris L.; Dias, Ana; Roque, Ana C. A.

doi:10.1007/978-1-0716-0775-6_10

Cited by 20 publications

(9 citation statements)

References 161 publications

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“…The modular protein T22-GFP-H6 has been used in this study as a model. T22 (red box) is a cationic peptide targeting CXCR4 [53], L (brown box) is a peptidic linker (GGRSSRSS) that provides flexibility, GFP (green box) is an enhanced GFP and H6 is a hexahistidine tag (black box) [54,55]. H6 is useful for both chromatographic purification of the protein [54][55][56][57] and cation-mediated assembly [55,58].…”

Section: Resultsmentioning

confidence: 99%

Time-Prolonged Release of Tumor-Targeted Protein–MMAE Nanoconjugates from Implantable Hybrid Materials

et al. 2022

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The sustained release of small, tumor-targeted cytotoxic drugs is an unmet need in cancer therapies, which usually rely on punctual administration regimens of non-targeted drugs. Here, we have developed a novel concept of protein–drug nanoconjugates, which are packaged as slow-releasing chemically hybrid depots and sustain a prolonged secretion of the therapeutic agent. For this, we covalently attached hydrophobic molecules (including the antitumoral drug Monomethyl Auristatin E) to a protein targeting a tumoral cell surface marker abundant in several human neoplasias, namely the cytokine receptor CXCR4. By this, a controlled aggregation of the complex is achieved, resulting in mechanically stable protein–drug microparticles. These materials, which are mimetics of bacterial inclusion bodies and of mammalian secretory granules, allow the slow leakage of fully functional conjugates at the nanoscale, both in vitro and in vivo. Upon subcutaneous administration in a mouse model of human CXCR4+ lymphoma, the protein–drug depots release nanoconjugates for at least 10 days, which accumulate in the tumor with a potent antitumoral effect. The modification of scaffold cell-targeted proteins by hydrophobic drug conjugation is then shown as a novel transversal platform for the design of slow releasing protein–drug depots, with potential application in a broad spectrum of clinical settings.

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Section: Resultsmentioning

confidence: 99%

Time-Prolonged Release of Tumor-Targeted Protein–MMAE Nanoconjugates from Implantable Hybrid Materials

et al. 2022

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“…9 Common epitope tags such as His, Myc, HA, and Flag have been reviewed extensively. 10,11 The first use of epitope tagging was described by Munro and Pelham in 1984, 12 followed shortly thereafter by the introduction of the Flag tag, 13 HA tag, 14 His tag, 15 and Myc tag. 16 Since then, many additional affinity tags have been designed and developed.…”

Section: Discussionmentioning

confidence: 99%

“…The plasmid pGEX-5X1-C-MBP 1-39 was constructed by combining a PCR fragment encoding amino acids 1-19 of MBP produced using primers 3 and 4 and pMAL-vc5X as a template with a PCR of pGEX-5X1 (GE Life Sciences) using primers 5 and 6 using NEBuilder HiFi DNA assembly (NEB Cat#E5520). Truncations of the resulting plasmid were made using the Q5 Site-directed mutagenesis kit and primer 6 plus primer (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). Helix-promoting and hydrophilicity mutations G19A, L20R, V23L, and G24A were added to pGEX-5X1-MBP 1-39 by assembling a pGEX-5X1 PCR fragment using primers 6 and 18 with oligo 2, a duplex g-block (IDT) using NEBuilder HiFi DNA assembly.…”

Section: Construction Of Gst-mbp Plasmidsmentioning

confidence: 99%

A useful epitope tag derived from maltose binding protein

Lénon

Ren

et al. 2021

Protein Science

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Maltose binding protein (MBP) is used in recombinant protein expression as an affinity and solubility tag. The monoclonal antibody B48 binds MBP tightly and has no cross-reactivity to other proteins in an Escherichia coli lysate. This high level of specificity suggested that MBP contains an epitope that could prove useful as a purification and visualization tag for proteins expressed in E. coli. To discover the MBP epitope, a co-crystal structure was determined for MBP bound to its antibody and four amino acids of MBP were identified as critical for the binding interaction. Fusions of various fragments of MBP to the glutathione S-transferase protein were engineered in order to identify the smallest fragment still recognized by the α-MBP antibody. Stabilization of the epitope via mutational engineering resulted in a minimized 14 amino-acid tag.

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“…signal sequences to accumulate into specific subcellular compartments such as apoplast, chloroplast, and endoplasmic reticulum ( Habibi et al., 2017 ). Recombinant proteins can subsequently be purified with affinity tags such as maltose-binding protein, glutathione S-transferase (GST), thioredoxin, staphylococcal protein A, and poly-histidine tag ( Pina et al., 2021 ).…”

Section: Heterologous Expression Of Recombinant Proteins In Plantsmentioning

confidence: 99%

Plant-based expression platforms to produce high-value metabolites and proteins

Kulshreshtha¹,

Sharma

Padilla³

et al. 2022

Front. Plant Sci.

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Plant-based heterologous expression systems can be leveraged to produce high-value therapeutics, industrially important proteins, metabolites, and bioproducts. The production can be scaled up, free from pathogen contamination, and offer post-translational modifications to synthesize complex proteins. With advancements in molecular techniques, transgenics, CRISPR/Cas9 system, plant cell, tissue, and organ culture, significant progress has been made to increase the expression of recombinant proteins and important metabolites in plants. Methods are also available to stabilize RNA transcripts, optimize protein translation, engineer proteins for their stability, and target proteins to subcellular locations best suited for their accumulation. This mini-review focuses on recent advancements to enhance the production of high-value metabolites and proteins necessary for therapeutic applications using plants as bio-factories.

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Affinity Tags in Protein Purification and Peptide Enrichment: An Overview

Cited by 20 publications

References 161 publications

Time-Prolonged Release of Tumor-Targeted Protein–MMAE Nanoconjugates from Implantable Hybrid Materials

Time-Prolonged Release of Tumor-Targeted Protein–MMAE Nanoconjugates from Implantable Hybrid Materials

A useful epitope tag derived from maltose binding protein

Plant-based expression platforms to produce high-value metabolites and proteins

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