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Related Concept Videos

Tagging and Fusion Proteins01:24

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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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Immunoprecipitation, or IP, is a widely used technique that employs protein-antibody interactions to isolate proteins or protein complexes in their native state for studying protein-protein interactions, quaternary structures, or supramolecular complexes. Various modifications of the technique, including chromatin IP, cross-linking IP, and fluorescence IP, are commonly used.
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Related Experiment Video

Updated: Apr 9, 2026

Author Spotlight: Optimizing Affinity Chromatography for His-Tagged FEN1 Protein
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Protein Affinity Purification using Intein/Chitin Binding Protein Tags.

Sarah F Mitchell1, Jon R Lorsch2

  • 1Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

Methods in Enzymology
|June 23, 2015
PubMed
Summary
This summary is machine-generated.

This study presents a one-step method for purifying untagged recombinant proteins using a self-cleaving intein and chitin-binding domain (CBD). This approach avoids leaving tags on the protein or requiring additional purification steps.

Keywords:
CBDCell harvestingCell lysisChitin beadsIMPACT™-SystemIntein/Chitin binding protein tagsProtein affinity purification

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Protein Chemistry

Background:

  • High-purity recombinant protein isolation is crucial for biochemical and biophysical assays.
  • Existing affinity purification methods often result in tagged proteins or require additional protease removal steps.

Purpose of the Study:

  • To develop a one-step purification protocol for untagged recombinant proteins.
  • To overcome the limitations of traditional affinity purification methods.

Main Methods:

  • Utilized a fusion protein strategy incorporating a self-cleaving intein and a chitin-binding domain (CBD).
  • Employed thiol-catalyzed cleavage for intein removal.
  • Leveraged chitin affinity chromatography for purification.

Main Results:

  • Achieved one-step chromatographic purification of untagged proteins.
  • Demonstrated highly specific affinity purification.
  • Yielded pure protein free from N- or C-terminal extensions.

Conclusions:

  • The intein-CBD fusion system provides an efficient method for obtaining highly pure, untagged recombinant proteins.
  • This protocol simplifies protein purification and eliminates the need for post-purification tag removal.