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

PLoS One. 2017 Jul 11;12(7):e0181186. doi: 10.1371/journal.pone.0181186. eCollection 2017.


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.

MeSH terms

  • Animals
  • Copepoda / genetics
  • Copepoda / metabolism*
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism*
  • HCT116 Cells
  • Humans
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism
  • Mutation / genetics


  • Luminescent Proteins
  • Green Fluorescent Proteins

Grants and funding

NEC Solution Innovators provided support in the form of salaries for IS, AS, KH, and WI, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.