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The high photostability and brightness of StayGold allowed us to select moderately bright cells for observation with continuous illumination and to perform experiments in which imaging performance was not limited by photobleaching.

C. uchidae (phylum Cnidaria, class Hydrozoa) produces colonies of fluorescent polyps on shells of Nassarius livescens, a gastropod living in the sandy-mud bottom of the sea. It also produces millimeter-sized free-swimming medusae that express green fluorescence in the epithelium of the ex-umbrella and the sub-umbrella, as well as in the gonads. Our RNA sequencing and subsequent anchored PCR analyses using total RNA from C. uchidae identified a transcript encoding an FP with a chromophore-forming tripeptide GYG at the appropriate position. This protein [is] referred to as CU17S.

CU17S has a unique primary structure. The closest homologue, sharing only 15.2% identity, was obeCFP, an FP cloned from Obelia medusa (Aglyamova et al. 2011)

StayGold’s head and tail are relatively short, which makes this FP sensitive to C- and N-terminal fusions. We borrowed termini from other proven FPs and found that nine amino acids from the N-terminal region of EGFP (n1 or n2) and ten amino acids at the C-terminus of dfGFP (c4) could be fused to StayGold to improve its targeting function to some subcellular components (Supplementary Fig. 9).

For protein fusion applications, we attempted to fuse two copies of a StayGold construct that was appended at both the N- and C-termini using a flexible linker (EV linker)29 to create a tandem dimer. The resultant FP construct, tdStayGold, showed the same photostability as StayGold. An OSER assay3 using CytERM-tdStayGold showed almost no whorl structures, indicating the monovalent fusion of tdStayGold.

StayGold’s head and tail are relatively short, which makes this FP sensitive to C- and N-terminal fusions. We borrowed termini from other proven FPs and found that nine amino acids from the N-terminal region of EGFP (n1 or n2) and ten amino acids at the C-terminus of dfGFP (c4) could be fused to StayGold to improve its targeting function to some subcellular components.

Our qualitative observation by wide-field (WF) microscopy suggested that, although CU17S did not fluoresce brightly in E. coli or mammalian cells, the fluorescence did not photobleach substantially. However, because it is generally assumed that there is an inverse correlation between the brightness and photostability of FPs, CU17S was not expected to improve brightness while maintaining excellent photostability. Nevertheless, through random mutagenesis of the CU17S gene, we discovered that the V168A mutation effectively improved the efficiency of both protein expression and chromophore maturation of the FP; CU17S/V168A was abundantly produced in bacteria.

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