Abstract
Split inteins are powerful tools for seamless ligation of synthetic split proteins. Yet, their use remains limited because the already intricate split site identification problem is often complicated by the requirement of extein junction sequences. To address this, we augment a mini-Mu transposon-based screening approach and devise the intein-assisted bisection mapping (IBM) method. IBM robustly reveals clusters of split sites on five proteins, converting them into AND or NAND logic gates. We further show that the use of inteins expands functional sequence space for splitting a protein. We also demonstrate the utility of our approach over rational inference of split sites from secondary structure alignment of homologous proteins, and that basal activities of highly active proteins can be mitigated by splitting them. Our work offers a generalizable and systematic route towards creating split protein-intein fusions for synthetic biology.
Highlights
Split inteins are powerful tools for seamless ligation of synthetic split proteins
This process is known as protein splicing and it produces a product as if the intein was absent from the original gene sequence
Whilst this shares some similarities with the search of a general split site, the presence of an intein introduces an extra layer of complexity—inteins require specific extein junction sequences for efficient splicing[19,20]
Summary
Split inteins are powerful tools for seamless ligation of synthetic split proteins. Yet, their use remains limited because the already intricate split site identification problem is often complicated by the requirement of extein junction sequences. An intein excises itself from the precursor protein and ligates the flanking external proteins (exteins) with a peptide bond This process is known as protein splicing and it produces a product as if the intein was absent from the original gene sequence. One method searches for flexible regions on protein structures and regions that lack functional conservation, and was demonstrated on inserting the gp[] intein on genes encoding antibiotic resistances[17,18] Another method abbreviated SPELL25 takes protein structures, calculates split energies and identifies surface-exposed loops that contains low conservation in sequences to predict split sites. SPELL was designed to split proteins with a pair of chemically inducible dimerization (CID) domains, and so might not be fully compatible with intein insertions While these computational methods provide better rationality in testing split sites, they rely on protein 3D structures. Testing only a few split sites risks missing the optimal sites, and this could jeopardize the overall performance of larger synthetic systems if they comprise split intein-inserted proteins as key components
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