Seleno-Biologics and 'Biobetters'
Site-specific incorporation of the 21st amino acid, selenocysteine (Sec), is of great interest for protein engineering because the selenol moiety has a lower pKa and superior nucleophilicity than a cysteine thiol. Pairs of selenocysteine residues can also form covalent diselenide bonds, an interesting structural motif with different properties than a disulfide bond. In nature, selenocysteine is biosynthesized directly on its tRNA from a precharged serine and incorporated in response to the opal (UGA) stop codon. This process requires an adjoining RNA element and unique protein factors to direct the bacterial translation machinery to reassign the stop codon. This RNA element forms part of the coding sequence and severely limits the use of selenocysteine as a tool for protein engineering. The process is also extremely inefficient and competes with termination of translation mediated by release factor 2 (RF2). Previously, we evolved an E. coli selenocysteinyl-tRNA (tRNASec) which is compatible with the canonical bacterial translation machinery and can incorporate selenocysteine with high efficiency in response to amber stop codons (UAG). Use of the UAG stop codon enables us to take advantage of recoded bacterial strains which lack the ability to use this codon as a termination signal, dramatically increasing incorporation efficiency. The disadvantage of this system is competing incorporation of serine by Ser-tRNASec (the biosynthetic precursor to selenocysteine), which can also participate in translation - a process prevented by SelB in nature.
There are many unanswered questions around selenocysteine biosynthesis and its unique incorporation pathway, and we are continually developing this technology with the goal of producing selenoprotein therapeutics containing diselenide bonds. These bonds are resistant to reduction and will not undergo rearrangement with free thiol groups, which can extend the effective half-life of therapeutics and improve efficacy. We have several interesting projects in this area (which we love talking about).