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Tagging and Fusion Proteins

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: Nov 28, 2025

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

Published on: December 9, 2016

Driving integrative structural modeling with serial capture affinity purification.

Xingyu Liu¹, Ying Zhang¹, Zhihui Wen¹

¹Stowers Institute for Medical Research, Kansas City, MO 64110.

Proceedings of the National Academy of Sciences of the United States of America

|December 1, 2020

Summary

This summary is machine-generated.

Serial capture affinity purification (SCAP) enables efficient characterization of protein complexes. This method combines purification, imaging, and mass spectrometry to build integrative structural models of protein interactions.

Keywords:

chromatin cross-linking mass spectrometry epigenetics integrative structural modeling quantitative imaging

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Last Updated: Nov 28, 2025

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Area of Science:

Biochemistry
Structural Biology
Molecular Systems Biology

Background:

Characterizing protein complexes is crucial for understanding cellular functions and networks.
Existing methods for protein complex analysis can be time-consuming and lack multi-faceted validation.
Developing streamlined techniques for protein interaction network studies is essential.

Purpose of the Study:

To introduce a novel method, serial capture affinity purification (SCAP), for streamlined protein complex characterization.
To demonstrate the utility of SCAP in validating protein interactions in vivo and generating integrative structural models.
To provide a comprehensive approach combining multiple techniques for detailed protein complex analysis.

Main Methods:

Serial Capture Affinity Purification (SCAP) using HaloTag and SNAP-tag for multistep enrichment.
In vivo interaction validation via acceptor photobleaching Förster resonance energy transfer (FRET) and fluorescence cross-correlation spectroscopy (FCCS).
Coupling SCAP with cross-linking mass spectrometry (XL-MS) for structural modeling.

Main Results:

SCAP successfully enabled multistep affinity enrichment of specific protein complexes.
In vivo imaging techniques confirmed protein interactions within live cells.
Integration of SCAP with XL-MS yielded an integrative structural model of the Spindlin1-SPINDOC complex.
The structural model revealed SPINDOC binding to the histone H3 interaction interface of SPIN1.

Conclusions:

SCAP offers a streamlined and versatile approach for characterizing protein complexes.
The combined methodology allows for in vivo validation and the generation of high-resolution structural models.
This integrated strategy advances the study of protein interaction networks and complex structures.