(27bf) Engineering High-Throughput Fluorescent Reporters for Selenocysteine Incorporation

Selenocysteine (Sec) is a rare but naturally occurring amino acid with a very similar structure to cysteine, except the thiol group is replaced with a selenol group. The higher nucleophilicity of selenium offers improved properties in selenoproteins such as higher activity in Sec mediated catalytic centers and greater stability of the protein structure by incorporating diselenide bonding pairs instead of disulfide bonding pairs. A major bottleneck to exploiting these properties is the development of efficient pathways for synthesizing and incorporating Sec into proteins, many elements of which have been identified but require further study and engineering. Such work necessitates the use of high-throughput reporters directly dependent on the correct and efficient incorporation of Sec. We have previously demonstrated the utility of small ultra-red fluorescent protein (SmURFP) as a Sec reporter. SmURFP (Ex 642 nm/Em 670 nm) covalently binds biliverdin as a co-factor chromophore at a cysteine residue making the protein dependent on the thiol chemistry to yield a fluorescence signal. By substituting the cysteine codon with a UAG stop codon and co-expressing a variant tRNA^Sec which functions as a UAG suppressor, along with the other selenocysteine biosynthesis and incorporation machinery, we can convert SmURFP into a Sec-dependent reporter. Unfortunately, like other cofactor-dependent far red fluorescent proteins, smURFP displays relatively low brightness and long maturation times compared to fluorescent proteins derived from the handful of highly engineered beta barrel scaffolds.

We sought to engineer an improved smURFP reporter using a Deep Mutational Scanning (DMS) strategy in combination with high-throughput screening. A dual plasmid circuit was developed with one plasmid expressing wild-type smURFP under the control of a tetracycline inducible promoter (plTetO) and the cognate TetR repressor, and the other plasmid constitutively expressing Synechocystis heme oxygenase 1 (SynHO-1) which is used to generate the biliverdin cofactor in excess. A DMS library was built from oligo pools designed to encode every single amino acid substitution, as well as single deletions and insertions throughout the entire 134 residue protein (5628 library members). The library was expressed in E. coli strain DH10B and sorted via FACS to isolate the most fluorescent clones. Combinatorial assembly of the identified single mutations using DNA shuffling is ongoing and expected to yield significant improvements over the parental sequence. We anticipate that these improved smURFP variants will find broader utility as both high-throughput genetic reporters for selenocysteine incorporation and additional imaging applications in eukaryotic cells.