Affinity Tags for Protein Purification

Mishra, Vibhor

doi:10.2174/1389203721666200606220109

Cited by 35 publications

(31 citation statements)

References 79 publications

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“…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]. N and C indicate the amino and carboxy-terminal ends, respectively.…”

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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“…The epitope tags are usually small peptides, including Flag, HA, V5, Myc, Strep, and His. And protein/domain tags contain GST, MBP, SUMO, CBP, Halo, Nus A, and FATT [33]. His tag and Strep-tag are the most commonly used affinity tags in protein ubiquitination profiling [1,8].…”

Section: Insights Into Ubiquitinated Proteins Using Ub Tagging-based ...mentioning

confidence: 99%

Current methodologies in protein ubiquitination characterization: from ubiquitinated protein to ubiquitin chain architecture

Sun

Zhang

2022

Cell Biosci

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Ubiquitination is a versatile post-translational modification (PTM), which regulates diverse fundamental features of protein substrates, including stability, activity, and localization. Unsurprisingly, dysregulation of the complex interaction between ubiquitination and deubiquitination leads to many pathologies, such as cancer and neurodegenerative diseases. The versatility of ubiquitination is a result of the complexity of ubiquitin (Ub) conjugates, ranging from a single Ub monomer to Ub polymers with different length and linkage types. To further understand the molecular mechanism of ubiquitination signaling, innovative strategies are needed to characterize the ubiquitination sites, the linkage type, and the length of Ub chain. With advances in chemical biology tools, computational methodologies, and mass spectrometry, protein ubiquitination sites and their Ub chain architecture have been extensively revealed. The obtained information on protein ubiquitination helps to crack the molecular mechanism of ubiquitination in numerous pathologies. In this review, we summarize the recent advances in protein ubiquitination analysis to gain updated knowledge in this field. In addition, the current and future challenges and barriers are also reviewed and discussed.

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“…Column affinity chromatography in which drugs are coupled to matrices have been used to identify drug-binding proteins (Jones et al, 2015;Katiyar et al, 2013;Mercer et al, 2011;Rylova et al, 2015;Schenone et al, 2013;Shi et al, 2012;Thomas et al, 2017). Here it is important to remember that proteins eluted from the column, even after rigid washing protocols (Amarasinghe and Jin, 2015;Mensa-Wilmot et al, 1995;Mishra, 2020), might not bind the drug that is coupled to the chromatography matrix; some eluted proteins may be present on the column due to interaction with other proteins that bound directly to the drug. For this reason, a second set of studies that measure direct interactions between drugs and proteins (e.g., surface plasmon resonance (Douzi, 2017;Nikolovska-Coleska, 2015); microscale thermophoresis (Hellinen et al, 2020;Khavrutskii et al, 2013;Wienken et al, 2010); thermal shift assays (Alshareef et al, 2016;Dziekan et al, 2019;Lucet and Murphy, 2017;Martinez Molina et al, 2013); activity-based proteomic profiling (Cravatt et al, 2008;Golkowski et al, 2017) or drug inhibition of purified enzyme activity (Guyett et al, 2016;Labar et al, 2007;Simcic et al, 2014;Warrilow et al, 2016) is essential for confirming drug-binding properties of proteins identified with affinity chromatography techniques.…”

Section: Unbiased Strategies For Discovery Of Drug-binding Proteins In Cellsmentioning

confidence: 99%

How Physiological Targets Can Be Distinguished from Drug-binding Proteins

Mensa-Wilmot

2021

Mol Pharmacol

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Some drugs in clinical trial owe their effectiveness to "off-target" activity. This and other observations raise a possibility that many studies to identify targets of drugs are incomplete. If "off-target" proteins are pharmacologically important it will be worthwhile to identify them early in the development process, for a better understanding of the molecular basis of drug action. Herein, we outline a multidisciplinary strategy for systematic identification of physiological targets of drugs in cells. A drugbinding protein whose genetic disruption yields very similar molecular effects as treatment of cells with the drug may be defined as a physiological target of the drug. For a drug developed with a "rational approach", it is desirable to verify experimentally that a protein used for hit optimization in vitro remains the sole polypeptide recognized by the drug in a cell.

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Affinity Tags for Protein Purification

Cited by 35 publications

References 79 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

Current methodologies in protein ubiquitination characterization: from ubiquitinated protein to ubiquitin chain architecture

How Physiological Targets Can Be Distinguished from Drug-binding Proteins

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