Cloning and Expression of the Green Fluorescent Protein (GvFP) from the Clinging Jellyfish (Gonionemus vertens) Using RNA-Seq
Date of Award
Master of Science (MS)
College of Science and Mathematics
Thesis Sponsor/Dissertation Chair/Project Chair
John J. Gaynor
Paul A.X. Bologna
We have discovered a novel GFP in the clinging jellyfish (Gonionemus vertens), a protein we have named GvFP (Gonionemus vertens Fluorescent Protein). This protein is responsible for the bright green fluorescence seen in the rhopalia along the margin of the bell, the gonads associated with the radial canals, and the manubrium when illuminated with blue light (470 nm). GvFP was first identified by Nextgen sequencing (RNA-Seq) of libraries made from mRNA of mature medusa collected from a salt pond on Martha’s Vineyard, MA. The full-length GvFP gene is 690 bp, encoding a protein of 230 AA (25.95 kDa; pI = 5.74). This protein shows low homology (43%) to the classic wtGFP from Aequorea victoria, but higher homology (86%) to another GFP (ScSuFP) from the Hydrozoan Scolionema suvaence. The tripeptide chromophore in GvFP - MYG - is unique among hydrozoans and has only been reported once before in Anemonia sulcata (Class Anthozoa). PHYRE2 analysis of GvFP predicts a β-barrel conformation and a 2° structure of 55% β-sheet, 7% alpha-helix, and 13% disordered structure, modelled at >90% confidence. The dimensions of the protein are 6.8 nm x 5.4 nm x 3.8 nm. Cloning of the full-length GvFP (Contig 6770) into the pET-SUMO expression vector and transformation into E. coli (BL21(DE3)) has expressed a recombinant GvFP that is a near perfect match in excitation and emission maxima of the native in vivo GvFP. Future work on this novel GFP may shed light on its unique chromophore and further our understanding of the ecological consequences of biological fluorescence in this jellyfish.
Orologas, Vasilios A., "Cloning and Expression of the Green Fluorescent Protein (GvFP) from the Clinging Jellyfish (Gonionemus vertens) Using RNA-Seq" (2020). Theses, Dissertations and Culminating Projects. 324.