Protein Splicing Mechanisms and Applications

Protein splicing is a self-catalysed post-translational process in which an intervening polypeptide segment, known as an intein, excises itself from a precursor protein and ligates the flanking sequences (exteins) to yield a mature protein product. This reaction proceeds via a series of nucleophilic displacement steps that may occur within a single polypeptide chain (cis-splicing) or between separate fragments (trans-splicing). Split inteins, in particular, enable the seamless assembly of protein segments from distinct precursors, facilitating scarless ligation and precise control of protein architecture. Advances in intein engineering have yielded variants with enhanced reaction rates, broadened extein compatibility and conditional activation, underpinning diverse applications. These include site-specific labelling, protein cyclisation, biosensor development, modular protein assembly and non-chromatographic purification. The ability to programme protein connectivity at the amino-acid level has transformed approaches in synthetic biology, biomedical imaging and industrial biotechnology, offering global significance through tailored biocatalysts and dynamic regulatory systems.

Research from Nature Portfolio

Recent studies have described the rational design of a cysteine-less split intein that overcomes oxidative sensitivity and aggregation-mediated inactivity. By mapping aggregation-prone regions and engineering monomeric mutants, researchers achieved nearly quantitative splicing efficiency while retaining an ultra-fast reaction rate. This optimised intein operates independently of thiol chemistry, expanding the toolkit for oxidative environments and orthogonal labelling strategies.

Another report introduced an expanded library of orthogonal split inteins characterised by high mutual independence and splicing fidelity. Through systematic screening of over thirty intein candidates, fifteen orthogonal pairs were validated for in vivo logic circuit construction and in vitro assembly of large repetitive proteins. This resource enables multiplexed protein engineering, permitting simultaneous, scarless ligation of multiple polypeptide segments for complex synthetic biology applications.

Protein Splicing Mechanisms and Applications publication trend

The graph below shows the total number of articles in protein splicing mechanisms and applications across all publications each year (not limited to Nature Index journals).

Technical terms

Intein: An intervening protein segment that self-excises and catalyses ligation of flanking sequences.

Extein: The N- and C-terminal sequences that are ligated together following intein excision.

Cis-splicing: Protein splicing occurring within a single contiguous polypeptide chain.

Trans-splicing: Protein splicing between two separate polypeptide fragments, enabling modular protein assembly.

Orthogonal intein: An intein pair that splices specifically with its partner without cross-reacting with other inteins.

Protein trans-splicing: The intein-mediated ligation of two extein segments derived from distinct precursor polypeptides.

References

1. Real‐Time, Non‐Invasive Monitoring of Neuronal Differentiation Using Intein‐Enabled Fluorescence Signal Translocation in Genetically Encoded Stem Cell‐Based Biosensors. Advanced Functional Materials (2024).
2. A cysteine-less and ultra-fast split intein rationally engineered from being aggregation-prone to highly efficient in protein trans-splicing. Nature Communications (2025).
3. An expanded library of orthogonal split inteins enables modular multi-peptide assemblies. Nature Communications (2020).
4. Unprecedented Rates and Efficiencies Revealed for New Natural Split Inteins from Metagenomic Sources*. Journal of Biological Chemistry (2012).
5. Structural Basis of the Bivalency of the TRPV1 Agonist DkTx. Angewandte Chemie International Edition (2023).