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Tagging and Fusion Proteins

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Proteins are involved in several cellular processes and biochemical reactions. Analyzing a specific protein of interest requires it to be isolated from the other proteins in the cell. This is achieved by overexpressing the specific gene in a suitable host to produce large quantities of the target protein. A tag or label is recombined with the gene to produce a fusion protein containing the target protein and the tag. The tags on these fusion proteins can then be used for easy detection and...

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Updated: Sep 19, 2025

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

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Published on: November 12, 2012

Purification tags markedly affect self-aggregation of CPEB3.

Harunobu Saito¹, Yujin Lee¹, Motoharu Ueno²

¹Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Japan.

|June 17, 2025

Summary

Purification tags on recombinant proteins can alter protein aggregation, including liquid-liquid phase separation and amyloid formation. Researchers must account for tag effects for accurate results in protein aggregation studies.

Keywords:

CPEB3 His tag IDR LLPS amyloid fibril protein aggregation

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Last Updated: Sep 19, 2025

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Published on: November 12, 2012

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Published on: June 10, 2020

Area of Science:

Biochemistry and Molecular Biology
Protein Science
Biophysical Chemistry

Background:

Protein aggregation, encompassing liquid-liquid phase separation (LLPS) and amyloid fibril formation, is crucial in biological functions and diseases.
In vitro studies using recombinant proteins are vital for understanding protein aggregation mechanisms.
The presence of uncleaved purification tags can potentially interfere with experimental outcomes.

Purpose of the Study:

To investigate the impact of common purification tags (His₆-GFP and His₁₂) on protein aggregation.
To determine if these tags influence the liquid droplet and amyloid fibril formation of specific protein regions.

Main Methods:

Utilized recombinant mouse cytoplasmic polyadenylation element-binding protein 3 (CPEB3) intrinsically disordered region (IDR) and its fragments.
Employed in vitro assays to assess liquid droplet formation and amyloid fibril formation.
Compared aggregation behavior with and without His₆-GFP and His₁₂ purification tags.

Main Results:

His₆-GFP and His₁₂ tags significantly affected both liquid droplet and amyloid fibril formation of CPEB3 IDR and its fragments.
The presence of purification tags altered the aggregation propensity and characteristics of the studied protein segments.

Conclusions:

Purification tags can substantially influence in vitro protein aggregation assays.
It is essential to consider and account for the effects of purification tags when interpreting results from protein aggregation studies.
Accurate interpretation of protein aggregation mechanisms requires careful evaluation of tag-protein interactions.