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

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.