Optical properties of the monomeric red fluorescent protein mRFP1

Abstract

For the expression in E. coli, the PCR-amplified fragment encoding mRFP1 from vector pMT-mRFP1 (Fungal Genetic Stock Center) was placed in the pQE-60 vector. Chemically competent E. coli ER1821 were transformed and grown overnight at 37°C. The protein was purified by Ni-NTA chromatography and dialyzed against 67mM Na2HPO4, 67mM KH2PO4, pH 7.5. There are two peaks (at 503 and 584 nm) in the mRFP1 absorption spectrum. The green component (503 nm) may correspond to a green fraction of the protein (a fraction that never matures beyond the green intermediate or a fraction which is trapped as a dead-end product such as the nonproductive trans conformation for the F65-Q66 peptide bond). The mRFP1’s extinction coefficient is equal to 42 mM−1 cm−1 at 584 nm; the emission maximum is at 607 nm; the excitation maximum is at 584–586 nm; the Stokes shift is equal to 23 nm; the fluorescence lifetime is about 1.8 ns; the quantum yield is 0.27; pKa is 4.0. Analysis of the mRFP1 absorption spectrum by high-order derivative spectroscopy shows that electron transition systems of both the fully matured form (absorption maximum at 584 nm) and the green fraction of the protein (absorption maximum at 503 nm) are practically identical.