|EC (M-1 cm-1)
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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.
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).
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.