Multi-Purpose Hyper-Stable Fluorescent Proteins

Background & Unmet Need

• Fluorescent proteins (FPs) are used for a broad range of biology research applications, including as reporters of gene activity and to visualize proteins in live cells
• Most FPs lack the ability to withstand the adverse conditions in the cell that rapidly denature biological structures, including superfolder GFP
• Modern microscopy techniques such as CLEM and ExM, place even greater demands on FPs due to secondary fixation and staining chemicals such as osmium tetroxide that degrade protein functionality
• Most widely used FPs are susceptible to misfolding due to exposure to higher temperatures for a greater lengths of time and photo bleach quickly
• Unmet Need: There is a need for stable FPs that can withstand chaotropic conditions and may be used for various biochemical applications

Technology Overview

• The Technology: Development of extremely stable FPs that withstand chaotropic conditions
• The engineered FPs Hyperfolder YFP (hfYFP) and LSSmGFP successfully survive CLEM sample preparation and ExM techniques
• hfYFP contains no cysteines, is chloride insensitive, and tolerates aldehyde and osmium tetroxide fixation better than common FPs
• The robust nature of hfYFP and LSSmGFP make them ideal FPs for the development of biosensors
• PoC Data: The chemical stability of hfYFP was employed to devise a fluorescence-assisted protein purification strategy, enabling all steps of denaturing affinity chromatography to be visualized using UV or blue light

Technology Applications

• Fluorescence-assisted protein purification
• Live cell imaging and the development of improved biosensors
• Stable FPs for use with CLEM and ExM techniques

Technology Advantages

• Compatible with osmium tetroxide and aldehyde fixation protocols
• Chloride insensitive
• Improved acid resistance
• Greater thermodynamic stability and faster refolding than existing FPs