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Related Experiment Video

Updated: Jan 14, 2026

Synthesis of an Intein-mediated Artificial Protein Hydrogel

Synthesis of an Intein-mediated Artificial Protein Hydrogel

Published on: January 27, 2014

Three-Segment Protein Labeling Using a Highly Efficient and Cysteine-Less Split Intein Identified with Computational

Christoph Humberg¹, Jonas Kröger¹, Shmuel Pietrokovski²

¹Department of Chemistry and Pharmacy, Institute of Biochemistry, University of Münster, Corrensstraße 36, 48149, Münster, Germany.

Angewandte Chemie (International Ed. in English)

|October 24, 2025

Summary

This summary is machine-generated.

Researchers discovered a new computational method to find efficient split inteins for protein engineering. This accelerates the development of novel tools for assembling proteins, enabling new possibilities in biotechnology.

Keywords:

Click biology Protein labeling Protein ligation Protein splicing Thiol bioconjugation

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Area of Science:

Biochemistry
Protein Engineering
Computational Biology

Background:

Split inteins are crucial for protein engineering, enabling protein assembly.
Current methods for split intein discovery are slow and lack predictability.
Only one efficient cysteine-less split intein was previously available.

Purpose of the Study:

To develop a computational approach for discovering novel split inteins with high splicing efficiency.
To enable a new tandem protein trans-splicing (PTS) scheme for assembling proteins from three segments.
To expand protein engineering capabilities beyond thiol-dependent or oxidizing conditions.

Main Methods:

Computational sequence analysis to predict split intein efficiency.
Investigated the correlation between aggregation propensity and splicing efficiency.
Validated candidates using size-exclusion chromatography and biochemical assays.
Demonstrated application using a trimodular non-ribosomal peptide synthetase (NRPS).

Main Results:

Identified an inverse correlation between predicted aggregation propensity and splicing efficiency.
Discovered LCGC14, a second highly efficient cysteine-less split intein.
Demonstrated orthogonal use of LCGC14 with CLm intein for assembling three selectively labeled protein segments.
Successfully assembled a trimodular NRPS using the new PTS scheme.

Conclusions:

Computational prediction of split intein efficiency significantly streamlines discovery.
The availability of multiple cysteine-less split inteins expands protein engineering versatility.
This advancement facilitates complex protein assembly and labeling strategies.