The influence of commonly used tags on structural propensities and internal dynamics of peptides

Brauer, M. Mónica; Zich, Maria Theresia; Önder, Kamil; Müller, Norbert

doi:10.1007/s00706-019-02401-x

Cited by 10 publications

(7 citation statements)

References 45 publications

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“…39 Furthermore, a NMR based study has also revealed the influence of commonly used tags on structural propensities and internal dynamics of peptides. 40 Importantly, the attached fluorescent dye, as an inbuilt moiety in the polypeptide chain, may influence the inherent properties of the proteins including their native conformation, their aggregation kinetics, and the morphology of their amyloid assembly. 39,40 In contrast to these unavoidable complications linked to the covalent tagging of fluorophores, our method produces stable fluorescent amyloid nanofibers without altering the aggregation kinetics and the characteristic b-structure of the amyloids (Fig.…”

Section: Application Of Rho-b-incorporated Amyloids For Experiments I...mentioning

confidence: 99%

A robust yet simple method to generate fluorescent amyloid nanofibers

Prajapati¹,

Ansari²,

Yadav³

et al. 2023

J. Mater. Chem. B

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Section: Application Of Rho-b-incorporated Amyloids For Experiments I...mentioning

confidence: 99%

A robust yet simple method to generate fluorescent amyloid nanofibers

Prajapati¹,

Ansari²,

Yadav³

et al. 2023

J. Mater. Chem. B

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“…In this study a fully working chromatographic workflow was implemented, which allows real time monitoring of the loading, washing, and elution step. The degradation of the tag can be a result due to its super exposed nature or problems in the sequence which can lead to degradation by proteases [15, 50]. A degradation of the tag (26 kDa eGFP, 29 kDa GFP‐(RH)4) could be possible and would explain the additional band which can be seen in Figure 3A in the lysate (lane 2) and the purified fraction (lane 3).…”

Section: Resultsmentioning

confidence: 99%

Purification of a peptide tagged protein via an affinity chromatographic process with underivatized silica

Rauwolf

Steegmüller

Schwaminger

et al. 2021

Engineering in Life Sciences

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Silica is widely used for chromatography resins due to its high mechanical strength, column efficiency, easy manufacturing (i.e. controlled size and porosity), and low‐cost. Despite these positive attributes to silica, it is currently used as a backbone for chromatographic resins in biotechnological downstream processing. The aim of this study is to show how the octapeptide (RH)4 can be used as peptide tag for high‐purity protein purification on bare silica. The tag possesses a high affinity to deprotonated silanol groups because the tag's arginine groups interact with the surface via an ion pairing mechanism. A chromatographic workflow to purify GFP fused with (RH)4 could be implemented. Purities were determined by SDS‐PAGE and RP‐HPLC. The equilibrium binding capacity of the fusion protein GFP‐(RH)4 on silica is 450 mg/g and the dynamic binding capacity around 3 mg/mL. One‐step purification from clarified lysate achieved a purity of 93% and a recovery of 94%. Overloading the column enhances the purity to >95%. Static experiments with different buffers showed variability of the method making the system independent from buffer choice. Our designed peptide tag allows bare silica to be utilized in preparative chromatography for downstream bioprocessing; thus, providing a cost saving factor regarding expensive surface functionalization. Underivatized silica in combination with our (RH)4 peptide tag allows the purification of proteins, in all scales, without relying on complex resins.

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“…On the other hand, this assay has some limitations, in common with many widely-used autophagic flux assays, such as: the need for overexpression of the exogenous reporter, although potentially, CRISPR knock in technology could be used to overcome this [ 47 ]; the potential accumulation of TOLLES in lysosomes over time and the possibility of affecting lysosomal activity; the use of a bulky tag that can potentially affect protein function or induce aggregation [ 48 ]. Recently described Halo-tag probe-based assays for autophagic flux may circumvent the latter consideration to some degree, but the Halo-tag is still relatively bulky compared to small epitope tags, and these probes are relatively low throughput [ 45 ].…”

Section: Discussionmentioning

confidence: 99%

Signal-Retaining Autophagy Indicator as a Quantitative Imaging Method for ER-Phagy

Jiménez-Moreno

Salomo-Coll

Murphy

et al. 2023

Cells

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Autophagy is an intracellular lysosomal degradation pathway by which cytoplasmic cargoes are removed to maintain cellular homeostasis. Monitoring autophagy flux is crucial to understand the autophagy process and its biological significance. However, assays to measure autophagy flux are either complex, low throughput or not sensitive enough for reliable quantitative results. Recently, ER-phagy has emerged as a physiologically relevant pathway to maintain ER homeostasis but the process is poorly understood, highlighting the need for tools to monitor ER-phagy flux. In this study, we validate the use of the signal-retaining autophagy indicator (SRAI), a fixable fluorescent probe recently generated and described to detect mitophagy, as a versatile, sensitive and convenient probe for monitoring ER-phagy. This includes the study of either general selective degradation of the endoplasmic reticulum (ER-phagy) or individual forms of ER-phagy involving specific cargo receptors (e.g., FAM134B, FAM134C, TEX264 and CCPG1). Crucially, we present a detailed protocol for the quantification of autophagic flux using automated microscopy and high throughput analysis. Overall, this probe provides a reliable and convenient tool for the measurement of ER-phagy.

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The influence of commonly used tags on structural propensities and internal dynamics of peptides

Cited by 10 publications

References 45 publications

A robust yet simple method to generate fluorescent amyloid nanofibers

A robust yet simple method to generate fluorescent amyloid nanofibers

Purification of a peptide tagged protein via an affinity chromatographic process with underivatized silica

Signal-Retaining Autophagy Indicator as a Quantitative Imaging Method for ER-Phagy

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