Biochemical characterization of bacterially expressed fluorescent proteins

초록

Fluorescent proteins (FPs) are proteins that emit light of a specific color (emission light) when exposed to light of a specific wavelength (excitation light). These proteins belong to a structurally homologous class with naturally occurring chromophores derived from a sequence of a few amino acids within their polypeptide chains. We generated a series of bacterial expression vectors for various fluorescent proteins with N-terminal histidine tags. These vectors were used to purify the fluorescent proteins using nickel columns. The purified proteins were then utilized to determine important biochemical parameters, such as excitation wavelength, emission wavelength, extinction coefficient, quantum yield, brightness, maturation time, and pKa. Quinine sulfate, fluorescein, and rhodamine B were used as references for quantum yield determination.