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Detection of Protein Aggregation using Fluorescence Correlation Spectroscopy
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The Effect of Protein Tagging on Aggregation and Phase Separation.

Harunobu Saito1, Kenji Sugase1

  • 1Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto, Japan.

Journal of Cellular Biochemistry
|June 15, 2026
PubMed
Summary

Protein tags, used in research, can significantly change how proteins assemble. This review highlights how tags affect protein aggregation and phase separation, urging researchers to treat them as variables, not neutral tools.

Keywords:
LLPSaggregationamyloid fibrilsbiomolecular condensatesintrinsically disordered proteinsprotein tagging

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Area of Science:

  • Biochemistry and Molecular Biology
  • Protein Biophysics
  • Structural Biology

Background:

  • Protein tags are essential tools for protein manipulation, including purification, solubilization, detection, and imaging.
  • However, these exogenous elements can interfere with the intrinsic self-assembly properties of proteins, especially intrinsically disordered proteins and low-complexity domains.

Purpose of the Study:

  • To review how various protein tags (affinity, solubility, fluorescent, chemical) influence protein aggregation, amyloid formation, and liquid-liquid phase separation (LLPS).
  • To classify the mechanisms by which tags perturb protein self-assembly.
  • To provide strategies for distinguishing intrinsic protein behavior from tag-induced artifacts.

Main Methods:

  • Literature review and classification of perturbation mechanisms.
  • Analysis of case studies across diverse protein systems (pathogenic amyloids, RNA-binding proteins, prions, etc.).
  • Discussion of experimental strategies for validation and interpretation.

Main Results:

  • Identified six primary categories of tag perturbation mechanisms: solubility enhancement, artificial multivalency, electrostatic interactions, local effects, metal coordination, and positional dependence.
  • Demonstrated that tags significantly alter assembly kinetics, phase boundaries, material properties, fibril morphology, oligomeric states, and observed phenotypes.
  • Observed that tags can either suppress or promote protein self-assembly, leading to non-native condensations or stabilization of alternative aggregate states.

Conclusions:

  • Protein tags are not inert tools but active variables that profoundly shape protein self-assembly states.
  • Experimental design must account for tag-induced effects to accurately study intrinsic protein behavior.
  • Careful interpretation of results and orthogonal validation methods are crucial for distinguishing intrinsic versus construct-dependent phenomena.