Subunits of highly fluorescent protein R-phycoerythrin as probes for cell imaging and single-molecule detection

R-phycoerythrin (R-PE) subunits and enzymatic digests were characterized by high-performance liquid chromatography (HPLC), capillary and gel electrophoresis, and HPLC-electrospray ionization mass spectrometry. Subunits were isolated from R-PE by HPLC and detected as single molecules by total internal reflection fluorescence microscopy (TIRFM). Favorable spectroscopic characteristics of R-PE subunits and digest peptides in the visible region of spectrum originate from phycoerythrobilin (PEB) and phycourobilin (PUB) chromophores. High absorption coefficients and fluorescence (even under denaturing conditions), broad excitation and emission fluorescence spectra, and low molecular weights make these molecules suitable for fluorescence labeling of biomolecules and cells.;Fluorescent proteins were further formed both in vitro and in vivo by attachment of PEB to recombinant apo-subunits of R-PE and their genetic fusions to maltose binding protein (MBP). Apo-alpha and apo-beta R-PE subunits were cloned from red algae Polisiphonia boldii into bacterium Escherichia coli (E. coli). Although expressed apo-subunits formed inclusion bodies, fluorescent holo-subunits were formed after incubation of E. coli cells with PEB. Holo-subunits contained both PEB and urobilin (UB) chromophores. Fluorescence and differential interference contrast (DIC) microscopy localized holo-subunits at poles of E. coli cells. Proteins formed by attachment of PEB to MBP-subunit fusions were soluble, displayed high fluorescence, contained only PEB, and were located either throughout cells or at cell poles. As measured by flow cytometry, cells containing fluorescent holo-subunits or MBP-subunit fusions were up to ten times brighter than control cells. Proteins formed by attachment of PEB to R-PE apo-subunits can be used as florescence reporters of gene expression and protein localization in cells, and in flow cytometry.;Finally, a high-throughput method was demonstrated which recorded emission fluorescence spectra of individual E. coli cells containing fluorescent proteins. Upon excitation with a 488 nm argon-ion laser many bacterial cells were imaged by a 20x microscope objective while they moved through a capillary tube. Fluorescence was dispersed by a transmission diffraction grating, and an intensified charge-coupled device (ICCD) camera recorded simultaneously the zero order and the first order spectrum for each cell. Demonstrated method could have a higher throughput, better sensitivity, and better spectral resolution compared to spectral flow cytometry.