Search research articles

ABOUT JoVE

Overview Leadership Blog JoVE Help Center

AUTHORS

Publishing Process Editorial Board Scope & Policies Peer Review FAQ Submit

LIBRARIANS

Testimonials Subscriptions Access Resources Library Advisory Board FAQ

RESEARCH

JoVE Journal Methods Collections JoVE Encyclopedia of Experiments Archive

EDUCATION

JoVE Core JoVE Business JoVE Science Education JoVE Lab Manual Faculty Resource Center Faculty Site

Terms & Conditions of Use

Related Concept Videos

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...

Conservation of Protein Domains Over Different Proteins

Conservation of Protein Domains Over Different Proteins

Protein domains are small structurally independent units that are part of a single amino acid chain. Although these domains are often structurally independent, they may rely on synergistic effects to perform their functions as part of a larger protein. Protein domains may be conserved within the same organism, as well as across different organisms.
A limited set of protein domains often duplicate and recombine during evolution. These domains can be organized in different combinations to...

Conservation of Protein Domains

Conservation of Protein Domains

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by

Same author

The bis-salphen Zn(II) unit: a versatile building block for self-assembled heteroleptic coordination cages.

Nanoscale·2026

Same author

TRIM2 E3 ligase substrate discovery reveals zinc-mediated regulation of TMEM106B in the endolysosomal pathway.

EMBO reports·2026

Same author

Mechanistic insights into recruitment and regulation of the RNA helicase UPF1 in replication-dependent histone mRNA decay.

Nature communications·2026

Same author

Real-time tracking of mRNP complex assembly reveals various mechanisms that synergistically enhance translation repression.

Cell reports·2025

Same author

Structural polymorphism of α-synuclein fibrils alters the pathway of Hsc70-mediated disaggregation.

The EMBO journal·2025

Same author

The 76th Mosbacher Colloquium: AI-driven (r)evolution in structural biology and protein design.

Biological chemistry·2025

Search research articles

Related Experiment Video

Updated: Apr 4, 2026

Enhanced Sample Multiplexing of Tissues Using Combined Precursor Isotopic Labeling and Isobaric Tagging cPILOT

Enhanced Sample Multiplexing of Tissues Using Combined Precursor Isotopic Labeling and Isobaric Tagging cPILOT

Published on: May 1, 2017

Efficient segmental isotope labeling of multi-domain proteins using Sortase A.

Lee Freiburger^1,2, Miriam Sonntag^3,4, Janosch Hennig^5,6

¹Institute of Structural Biology, Helmholtz Zentrum München, 85764, Neuherberg, Germany. lee.freiburger@tum.de.

Journal of Biomolecular NMR

|August 31, 2015

Summary

We developed a new method for segmentally labeling large proteins using Sortase A, improving yields and simplifying purification for nuclear magnetic resonance (NMR) studies.

Keywords:

Multi-domain proteins Protein expression Protein ligation Segmental isotope labeling Sortase A

More Related Videos

Quantitative Proteomics Using Reductive Dimethylation for Stable Isotope Labeling

Quantitative Proteomics Using Reductive Dimethylation for Stable Isotope Labeling

Published on: July 1, 2014

Protease- and Acid-catalyzed Labeling Workflows Employing 18O-enriched Water

Protease- and Acid-catalyzed Labeling Workflows Employing 18O-enriched Water

Published on: February 20, 2013

Related Experiment Videos

Last Updated: Apr 4, 2026

Enhanced Sample Multiplexing of Tissues Using Combined Precursor Isotopic Labeling and Isobaric Tagging cPILOT

Enhanced Sample Multiplexing of Tissues Using Combined Precursor Isotopic Labeling and Isobaric Tagging cPILOT

Published on: May 1, 2017

Quantitative Proteomics Using Reductive Dimethylation for Stable Isotope Labeling

Quantitative Proteomics Using Reductive Dimethylation for Stable Isotope Labeling

Published on: July 1, 2014

Protease- and Acid-catalyzed Labeling Workflows Employing 18O-enriched Water

Protease- and Acid-catalyzed Labeling Workflows Employing 18O-enriched Water

Published on: February 20, 2013

Area of Science:

Biochemistry
Structural Biology
Biophysics

Background:

Nuclear magnetic resonance (NMR) studies of multi-domain protein complexes offer insights into molecular interactions and dynamics.
Isotope labeling is crucial for NMR but challenging for large proteins due to signal overlap.
Existing segmental labeling methods often require extensive optimization and yield poor ligation results.

Purpose of the Study:

To present an optimized strategy for segmental isotope labeling of multi-domain proteins.
To improve the efficiency and yield of protein ligation for biophysical studies.
To enable routine production of milligram quantities of segmentally labeled proteins.

Main Methods:

Utilized the S. aureus transpeptidase Sortase A for protein ligation.
Implemented efficient removal of cleaved peptide fragments via centrifugal filtration.
Employed a strategic design of cleavable and non-cleavable affinity tags for protein purification.

Main Results:

Achieved efficient segmental labeling of multi-domain proteins.
Demonstrated successful purification of labeled proteins using optimized affinity tags.
Enabled the routine production of milligram amounts of purified segmentally labeled protein.

Conclusions:

The optimized Sortase A-mediated strategy significantly improves segmental protein labeling.
This method facilitates the study of large protein complexes using NMR and other biophysical techniques.
The protocol is robust and suitable for routine production of labeled proteins.