OPTIMIZATION OF PROTEIN PURIFICATION PATHS FOR CLOVER-RUBY2 FLUORESCENT PROTEINS FOR IN VIVO KINETIC MODEL DEVELOPMENT
Chromophores are photoactive molecules that can be excited by light and return to ground state by fluorescence. When two different fluorescent proteins are tethered together, an excited donor can transfer energy to the acceptor via a process called Förster Resonance Energy Transfer, FRET, if certain conditions are met. FRET results in the acceptor becoming excited as well, leading to emission at a specific wavelength. The phenomenon is useful for several different experiments such as protease assays, in which cleavage of the protein linking the donor and acceptor abolishes FRET as the pair separates. This mechanism would prove useful when comparing affinity and turnover rates of proteases on the fluorescent protein labeled surface loops of G protein coupled receptors, GPCR, in future investigations. However, the accuracy of the data can be affected by protein interactions that can inflate or deflate detectable FRET. There has been little research in the different methods of purification and their effects on the purity and functionality of the fluorescent proteins. To determine the effects by different chromatography columns, different purification protocols were devised. In addition, several experiments on the fluorescent proteins alone were done to determine the factors such as light, temperature, salinity, and concentration that contributed towards degradation and aggregation. Results identified the most successful protocol. Application of a G-25 desalting column followed by an ion exchange and gel filtration columns produced an almost pure substrate and the most functional mRuby-2. Additionally, maturation for 6 hours in low salt conditions allowed for optimized mRuby-2. Maintaining concentrations of the FRET pair at 1.5μM to 2.5μM provided concise quantity control data. The protocol can be utilized for in vitro vs in vivo model, to accurately determine the effectiveness of enzymes on GPCR surface loop.
Fussell, Robert Charles