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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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Synthesis of an Intein-mediated Artificial Protein Hydrogel

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Generating Ultra-Fast Protein trans-Splicing of a Cysteine-Less and Semisynthetic Split Intein for Chemical Protein

Christoph Humberg¹, Tobias M E Terhorst¹, Tim Pasch¹

¹Department of Chemistry and Pharmacy, Institute of Biochemistry, University of Münster, Münster, Germany.

Chembiochem : a European Journal of Chemical Biology

|March 25, 2026

Summary

Optimized cysteine-less split inteins achieve ultra-fast protein trans-splicing (PTS) for efficient protein labeling and semisynthesis. This advancement enables rapid chemical modification under diverse conditions, expanding protein engineering capabilities.

Keywords:

click biology posttranslational modification protein engineering protein ligation protein semisynthesis

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Generation of Native, Untagged Huntingtin Exon1 Monomer and Fibrils Using a SUMO Fusion Strategy

Generation of Native, Untagged Huntingtin Exon1 Monomer and Fibrils Using a SUMO Fusion Strategy

Published on: June 27, 2018

Area of Science:

Biochemistry
Molecular Biology
Chemical Biology

Background:

Cysteine-less split inteins facilitate protein trans-splicing (PTS) under oxidizing conditions, compatible with chemical labeling and various cysteine redox states.
A need existed for rapidly splicing, cysteine-less split inteins with short precursors for efficient solid-phase peptide synthesis and semisynthesis.

Purpose of the Study:

To generate an optimized, ultra-fast splicing cysteine-less split intein variant.
To enable efficient protein semisynthesis using chemically synthesized short intein precursors.

Main Methods:

Optimization of the artificial split site and flanking extein residues in a split CL (cysteine-less) intein.
Chemical synthesis of a short (26 amino acid) N-terminal intein precursor fragment.
Demonstration of protein semisynthesis via ultra-fast N-terminal peptide tag transfer.

Main Results:

Achieved a nine- to 16-fold increase in splicing rate, reaching second-scale kinetics.
Successfully synthesized a short N-terminal precursor amenable to solid-phase synthesis.
Demonstrated efficient protein semisynthesis by labeling a protein's N-terminus with a fluorescent peptide tag.

Conclusions:

The optimized split CL intein offers ultra-fast splicing kinetics, comparable to leading split inteins.
This engineered intein facilitates efficient protein semisynthesis with short, chemically synthesized precursors.
The development expands the scope of chemical protein labeling and modification strategies.