Molecular evolution of versatile derivatives from a GFP-like protein in the marine copepod Chiridius poppei

Abstract

Fluorescent proteins are now indispensable tools in molecular research. They have also been adapted for a wide variety of uses in cases involving creative applications, including textiles, aquarium fish, and ornamental plants. Our colleagues have previously cloned a yellow GFP-like protein derived from the marine copepod Chiridius poppei (YGFP), and moreover, succeeded in generating transgenic flowers with clearly visible fluorescence, without the need for high-sensitivity imaging equipment. However, due to the low Stokes shift of YGFP (10 nm), it is difficult to separate emitted light of a labeled object from the light used for excitation; hence, limitations for various applications remain. In this study, which was aimed at developing YGFP mutants with increased Stokes shifts, we conducted stepwise molecular evolution experiments on YGFP by screening random mutations at three key amino acids, based on their fluorescent characteristics and structural stabilities, followed by optimization of their fluorescence output by DNA shuffling of the entire coding sequence. We successfully identified an eYGFPuv that had an excitation maximum in UV wavelengths and a 24-fold increase in fluorescence intensity compared to the previously reported YGFP mutant (H52D). In addition, eYGFPuv exhibited almost 9-fold higher fluorescence intensity compared to the commercially available GFPuv when expressed in human colon carcinoma HCT116 cells and without any differences in cytotoxicity. Thus, this novel mutant with the desirable characteristics of bright fluorescence, long Stokes shift, and low cytotoxity, may be particularly well suited to a variety of molecular and biological applications.

Generation of brilliant green fluorescent petunia plants by using a new and potent fluorescent protein transgene

“…Using our novel fluorescent genes, we have succeeded in generating P. hybrida plants exhibiting brilliant green fluorescence, which could be easily visualized at whole plant level without using any high sensitive imaging equipment. Because the excitation maximum of eYGFPuv is within the UV-A region (400 nm) 23 , the fluorescence of these transgenic plants was observed without any emission filter, which constitutes a crucial progress from previously reported fluorescent flowers 21 . Notably, we could obtain bright fluorescent images under unexceptional image acquisition conditions (≤2 s exposure; Supplementary Table S2 ).…”

“…1b ) 23 . For reference, the sequences, structure, and detailed fluorescence properties of each fluorescent protein were briefly reproduced in supplementary information (Supplementary Figure S1 and Supplementary Table S1 ) 23 . We first examined the intracellular localizations of eYGFPuv and eYGFP stably expressed in tobacco BY-2 cells and confirmed that the fluorescence of these FPs, as well as that of intact CpYGFP, was mainly observed in the cytoplasm with no preferential localization to the nucleus or vacuole (Supplementary Figure S2 ).…”

“…The fluorescence excitation and emission spectra of eYGFPuv was compared to that of eYGFP, which was also CpYGFP derivative showing the same fluorescence spectrum as CpYGFP (Fig. 1b ) 23 . For reference, the sequences, structure, and detailed fluorescence properties of each fluorescent protein were briefly reproduced in supplementary information (Supplementary Figure S1 and Supplementary Table S1 ) 23 .…”

“…The image acquisition condition was listed in Supplementary Table S2 . While eYGFPuv (theoretical molecular weight (MW): 25.9 kDa) forms dimers, commercial GFPuv (theoretical MW: 28.0 kDa) retains monomeric forms under normal physiological conditions 23 . The experiments were repeated twice with similar results, and one representative result is shown.…”

“…These fluorescent flowers should accelerate the application of FPs to ornamental purposes. To develop such flowers, we employed eYGFPuv , a new and potent fluorescent protein transgene, which was recently developed as a CpYGFP derivative exhibiting bright fluorescence with maximum excitation under UV wavelength (398 nm) and a long Stokes shift (104 nm) 23 . Furthermore, eYGFPuv uniquely kept stable fluorescence even under acidic conditions (pKa = 3.0), which were unusual compared to other FPs such as enhanced green fluorescent protein (EGFP) (pKa = 5.7) 23 .…”