Background
Selenocysteine (Sec), the 21st amino acid, offers unique redox and catalytic properties that are valuable for biocatalysis, therapeutic protein engineering, and redox biology. However, its use in synthetic biology and protein engineering has been restricted by the complexity of its natural biosynthetic machinery. Sec incorporation typically requires a specific mRNA hairpin structure (SECIS element), a dedicated elongation factor (SelB), and specialized tRNA (tRNA^Sec). These requirements limit insertion to native selenoprotein contexts and prevent the precise incorporation of Sec at arbitrary positions within heterologous proteins. Attempts to reengineer Sec insertion using general translation factors or modified SECIS elements have suffered from poor efficiency, limited flexibility, and unintended interactions with host systems.
Technology overview
This technology introduces SelB-v2, a redesigned bacterial elongation factor that enables SECIS-independent incorporation of selenocysteine at UAG codons. SelB-v2 was developed by truncating the C-terminal SECIS-binding domain (residues S488–K614) and introducing 13 adaptive mutations through error-prone PCR and functional selection. The resulting variant efficiently delivers Sec-tRNA^Sec to ribosomes in standard E. coli systems, as demonstrated using fluorescent protein reporters and mass spectrometry of Sec-labeled proteins. Importantly, the system remains orthogonal to the host’s native elongation factor EF-Tu, allowing precise Sec incorporation without disrupting canonical translation. Additionally, engineering of the amino acid binding pocket produced SelB-v2-Ser, which selectively incorporates serine, illustrating modular tunability of amino acid specificity.
Benefits
- Enables SECIS-independent selenocysteine incorporation at UAG codons
- Operates orthogonally to native translation machinery
- Supports high-efficiency expression in standard E. coli strains
- Allows programmable insertion of Sec or other nonstandard amino acids
- Tunable binding pocket enables expanded genetic code engineering
Applications
- Redox-active enzyme engineering
- Therapeutic protein development
- Site-specific labeling with Sec or serine
- Synthetic biology and genetic code expansion
- Biocatalysis and protein stabilization
Opportunity
- Removes key barriers to routine use of selenocysteine in engineered proteins
- Provides a generalizable platform for site-specific noncanonical amino acid incorporation
- Ideal for academic and industrial applications in protein engineering and synthetic biology
- Available for exclusive licensing
Intellectual property
PCT/US2025/030815
