A New Bright Green-Emitting Fluorescent Protein – Engineered Monomeric and Dimeric Forms

Document Content:

This research introduces a novel fluorescent protein, named vivid Verde fluorescent protein (VFP), isolated from the coral Cyphastrea microphthalma. VFP exhibits rapid maturation at 37 °C and emits bright green light. The study details the engineering of VFP into exclusively monomeric (mVFP1, mVFP) and dimeric (dVFP) forms. These variants demonstrate significantly enhanced brightness compared to EGFP, with mVFP1 and mVFP being at least twice as bright, and dVFP being even brighter. The research highlights the potential applications of these VFP variants in both in vitro and in vivo detection scenarios, offering improved sensitivity and versatility for biological imaging and molecular studies.

Table of Contents:

• Keywords
• Correspondence
• Note
• Structured digital abstract
• Introduction
• Abbreviations
• Results
• Sequence of the new FP and relation to other known FPs
• Oligomeric state of VFP
• Spectral properties of VFP and its variants
• Fluorescence correlation spectroscopy measurements of size and photostability
• Application of VFP variants as detection markers
• Application of mVFP as an in vivo marker
• Discussion
• Materials and methods
• Plasmid constructions and mutations
• Sequence alignment and homology modeling
• Recombinant protein expression
• Analytical gel-filtration chromatography
• Absorption spectroscopy
• Fluorescence spectroscopy
• FCS
• Photobleaching
• Western blot assays
• mRNA microinjection assay
• Acknowledgements
• References
• Supporting information