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Related Concept Videos

Tagging and Fusion Proteins01:24

Tagging and Fusion Proteins

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: May 21, 2026

One-step CRISPR-based Strategy for Endogenous Gene Tagging in Drosophila melanogaster
07:23

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Published on: January 26, 2024

Taking down the FLAG! How insect cell expression challenges an established tag-system.

Peter M Schmidt1, Lindsay G Sparrow, Rebecca M Attwood

  • 1CSIRO Materials Science and Engineering, Parkville, Victoria, Australia. Peter.Schmidt@CSL.com.au

Plos One
|June 16, 2012
PubMed
Summary
This summary is machine-generated.

A common FLAG-tag used for protein purification is surprisingly affected by tyrosine sulfation, a post-translational modification. This modification can significantly reduce the effectiveness of FLAG-tagged proteins for purification.

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Transient Expression and Cellular Localization of Recombinant Proteins in Cultured Insect Cells
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Published on: April 20, 2017

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Protein Engineering

Background:

  • The FLAG-tag is a widely used peptide sequence for recombinant protein identification and purification.
  • Its popularity stems from its small size, solubility, enterokinase cleavage site, and available high-affinity antibodies.
  • However, its universal applicability has not been fully investigated, especially concerning post-translational modifications.

Purpose of the Study:

  • To investigate potential post-translational modifications affecting the FLAG-tag's efficacy in insect cells.
  • To identify the specific modification and its impact on FLAG-tag recognition by anti-FLAG antibodies.
  • To assess the implications of this modification for the reliability of FLAG-tag based purification strategies.

Main Methods:

  • Expression of FLAG-tagged proteins in insect cells.
  • Analysis of protein modification using mass spectrometry.
  • Assessment of anti-FLAG antibody binding to modified and unmodified proteins.
  • Quantification of purified protein yield.

Main Results:

  • A post-translational modification was identified in secreted FLAG-tagged proteins in insect cells.
  • This modification was characterized as tyrosine sulfation on the tyrosine residue within the FLAG epitope (DYK).
  • Sulfation significantly reduced the binding affinity of anti-FLAG antibodies, decreasing accessible protein for purification to less than 20%.

Conclusions:

  • Tyrosine sulfation is a critical post-translational modification that can abolish FLAG-tag recognition.
  • This modification challenges the universal applicability of the FLAG-tag system for secreted proteins, particularly in insect cell expression systems.
  • Researchers should consider potential PTMs like sulfation when designing or utilizing FLAG-tagged proteins for purification.